Novel pyrimidine derivatives and methods of making and using these derivatives

ABSTRACT

This invention discloses pyrimidine derivatives, and pharmaceutically acceptable salts and prodrugs thereof, useful in therapeutically and/or prophylactically treating patients with an illness. Such illnesses include cancer, and secondary infections caused by  Pneumocystis carinii  and  Toxoplasmosis gondii  in immunocompromised patients. The compounds themselves, methods of making these compounds, and methods of using these compounds are all disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation-in-part application of U.S. applicationSer. No. 09/073,593, filed May 6, 1998, U.S. application Ser. No.09/190,374, filed Nov. 12, 1998, and U.S. application Ser. No.08/660,023 filed Jun. 6, 1996, all of which are hereby expresslyincorporated by reference.

[0002] This work was supported, in part, by grants from the NationalInstitute of General Medical Sciences GM52811(AG), and by the NationalInstitute of Allergy and Infectious Disease AI 41743 (AG).

FIELD OF THE INVENTION

[0003] This invention relates to pyrimidine derivative compounds andpharmaceutically acceptable salts thereof. More specifically, thisinvention relates to furo[2,3-d]pyrimidines, pyrrolo[2,3-d]pyrimidines,pyrrolo[3,2-d]pyrimidines, pyrrolo[3,4-d]pyrimidines,thieno[2,3-d]pyrimidines, cyclopentapyrimidines,cyclopenta[d]pyrimidines, pyrido[2,3-d]pyrimidines andpyrido[3,2-d]pyrimidines. “Pyrimidine derivatives” as used hereingenerally refers to all of these types of compounds. These compoundshave been found useful in resisting and treating Pneumocystis carinii,Toxoplasmosis gondii, Mycobacterium tuberculosis and Mycobacterium aviumcomplex (MAC) infections in immunocompromised patients, such as, forexample, patients with autoimmune deficiency syndrome (AIDS). Thesecompounds are also useful as potential antitumor, antituberculosis,anti-Mycobacterium avium, antibiotic, antimalarial, antifungal orantiprotozoal agents, or as synergistic agents when used withsulfonamides or other compounds and may require the use of leucovorinrescue. These compounds are also useful as antitumor agents in cancerpatients. Methods of preparing and using these compounds are alsoprovided.

BACKGROUND OF THE INVENTION

[0004] Various pyrimidine systems, such as the pyrido[2,3-d]pyrimidinering system, have been studied due to their involvement in theinhibition of dihydrofolate reductase (DHFR) enzymes activity. Thepyrimidine derivatives disclosed herein function as DHFR inhibitors.Because DHFR reduces dihydrofolate to tetrahydrofolate, inhibition ofDHFR deprives the cell of tetrahydrofolate, without which the cellcannot produce 5,10-methylenetetrahydrofolate.5,10-Methylene-tetrahydrofolate is essential for cell growth. Theinhibition of DHFR by the compounds, and pharmaceutically acceptablesalts thereof, of this invention therefore results in the inhibition ofDNA synthesis and leads to cell death. Methotrexate (MTX), trnetrexate(TMQ), piritrexim (PTX) and other folic acid analogues function asinhibitors of cell growth by similar mechanisms involving the inhibitionof dihydrofolate reductase.

[0005] The pyrimidine derivatives disclosed herein also function asthymidylate synthase (TS) inhibitors. TS, along with DHFR, forms part ofthe systems responsible for the synthesis of deoxythymidylate (dTMP)from deoxyuridylate (dUMP). TS catalyzes the sole de novo synthesis ofdTMP from dUMP. Inhibition of TS, therefore, deprives the cell ofthymidine, which is an essential constituent of DNA. Typically, thecompounds as described herein where X and Y are both NH₂ or where X isNH₂ and Y is H or CH₃ and will function as DHFR inhibitors, andcompounds where X is OH and Y is NH₂, H, or CH₃ will function as TSinhibitors, although the inventor does not wish to be bound by thisgenerality.

[0006] Drugs useful for the reduction of cancerous cells are also known.

[0007] Elslager, Edward F., et al., “Folate Antagonists. 20. Synthesisand Antitumor and Antimalarial Properties of Trimetrexate and Related6-[(Phenylamino)methyl]-2,4-quinazolinediamines” J. Med. Chem., Vol. 26pp. 1753-1760 (1983)), discloses the preparation of quinazolinediamines.This article states that the quinazolinediamines exhibit potentantimalarial, antibacterial and antitumor activity.

[0008] Methods of synthesizing diaminopyrido[2,3-d]pyrimidines havingvarious substituents are known. See Hurlbert, B. S., et al., “Studies onCondensed Pyrimidine Systems. XXIII. Synthesis of2,4-Diaminopyrido[2,3-d]pyrimidines from 6-Keto Esters”, J. Med. Chem.,Vol. 11, pp. 703-707 (1968), and Hurlbert, B. S., and Valenti, B. F.,“Studies on Condensed Pyrimidine Systems. XXIV. The Condensation of2,4,6-Triaminopyridimine with Malondialdehyde Derivatives”, J. Med.Chem., Vol. 11, pp. 708-710 (1968).

[0009] Hurlbert, B. S., et al., “Studies on Condensed PyrimidineSystems. XXV. 2,4-Diaminopyrido[2,3-d]pyrimidines. Biological Data”, J.Med. Chem., Vol. 11, pp. 711-717 (1968), discloses the antimicrobialactivities of several subgroups of pyridopyrimidines. This articlestates that 2,4-diaminopyrido[2,3-d]pyrimidines bearing alkyl andaralkyl substituents in the pyrimidine moiety are inhibitors ofdihydrofolate reductase having antibacterial and antiprotozoal activityand that these compounds potentiate sulfonamides.

[0010] Grivsky, E. M., et al., “Synthesis and Antitumor Activity of2,4-Diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3d]pyridimine”, J.Med. Chem., Vol. 23, pp. 327-329 (1980), discloses the synthesis of2,4-diamino-6-(2,5-dimethoxybenzyl)-5-methylpyrido[2,3-d]pyridimine(BW301U,7).This article states that BW301U,7 is as effective as methotrexate as aninhibitor of dihydrofolate reductase purified from human leukemic cellsand, in contrast to metoprine, has minimal activity as an inhibitor ofhistamine metabolism.

[0011] Shih et al., “LY231514, a Pyrrolo[2,3d]pyrimidine-basedAntifolate That Inhibits Multiple Folate-requiring Enzymes”, CancerResearch, Vol. 57, pp. 1116-1123 (1997) teaches a pyrrolo [2,3-d]pyrimidine-based antifolate that inhibits multiple folate-requiringenzymes. A classical or glutamic acid substituted pyrrolo pyrimidine isdisclosed.

[0012] Taylor et al., “A Dideazatetrahydrofolate Analogue Lacking aChiral Center at C-6,N-[4-[2-(2-Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl]-L-glutamicAcid, Is an Inhibitor of Thymidylate Synthase”, J. Med. Chem., Vol. 35,pp. 4450-4454 (1992) also teaches a classic pyrrolo pyrimidine compounduseful in the inhibition of thymidylate synthase. Taylor reports otherpyrrolo pyrimidine compounds in U.S. Pat. Nos. 4,996,206; 5,028,608;5,248,775; 5,254,687; and 5,344,932.

[0013] Werbel, Leslie, M., et al., “Synthesis and Antimalarial Activityof a S Series of 2,4-Diamino-6-[(N-alkylanilino)methyl]quinazolines[1,2]”, J. Heterocyclic Chem., Vol. 24, pp. 345-349 (1987), disclosesthe synthesis of N6 substituted quinazoline dihydrofolate reductaseinhibitors. This article states that these analogs demonstratesubstantial activity against Plasmodium berghei infections in mice.

[0014] Piper, J. R., et al., “Syntheses and Antifolate Activity of5-Methyl-5-deaza Analogues of Aminopterin, Methotrexate, Folic Acid, andN¹⁰-Methylfolic Acid”, J. Med. Chem., Vol. 29, pp. 1080-1087 (1986),discloses that 5-methyl-5-deaza analogues of aminopterin andmethotrexate are much more growth inhibitory than methotrexate.

[0015] Pyrido [2,3A and [3,2a pyrimidines are also disclosed in U.S.Pat. Nos. 5,346,900 and 5,508,281, and co-pending application Ser. Nos.08/515,491 and 08/660,023 all of which are hereby expressly incorporatedby reference.

[0016] Pyrrolo[2,3-d]pyrimidines are disclosed by Gangjee et al. in“Novel 2,4-diamino-5-substituted-pyrrolo[2,3-d]pyrimidines As Classicaland Non-Classical Antifolate Inhibitors of Dihydrofolate Reductases”, J.Med. Chem., Vol. 38, pp. 2158-2165 (Jun. 6, 1995).

[0017] Gangjee, A., et al., “Classical and Non-ClassicalFuro[2,3-d]Pyrimidines As Novel Antifolates: Synthesis and BiologicalActivities”, J. Med. Chem., Vol. 37, pp. 1169-1176 (1994), disclosesfuro[2,3-d]pyrimidines.

[0018] Mavandadi, et al., disclose 5-substituted classical andnonclassical 2,4-diaminopyrrolo[2,3-d]pyrimidines as antitoxoplasma,antipneuomocystis and antitumor agents in J. Med. Chem., 40:1173-1177(1997). Mavandadi, et al. also disclose use of pyrrolo[2,3-d]pyrimidinesas nonclassical inhibitors of thymidylate synthase in J. Med. Chem.,39:45634568 (1996).

[0019] There remains a very real and substantial need for compounds thatare more active and more selective than known compounds at resisting andtreating infections caused by Pneumocystis carinii and Toxoplasmosisgondii, and other organisms in immunocompromised patients, reducing thetumor size and/or the number of cancerous cells in cancer patients, andfor methods of preparing and using such compounds.

SUMMARY OF THE INVENTION

[0020] The present invention has met the above described need byproviding potent and selective pyrrolo[2,3-d]pyrimidine compounds andfuro[2,3-d]pyrimidine compounds.

[0021] Methods of synthesizing the above compounds are also disclosed.

[0022] This invention provides methods for therapeutically and/orprophylactically using the compounds, and pharmaceutically acceptablesalts and prodrugs thereof, described herein. More specifically, thisinvention provides methods of using the present pyrimidine derivativesfor therapeutic and prophylactic purposes including employing thesecompounds to resist and treat secondary infections caused byPneumocystis carinii, Toxoplasmosis gondii, Mycobacterium tuberculosisand Mycobacterium avium complex or other organisms in immunocompromisedpatients, such as for example patients with AIDS. The immunocompromisedpatient has a primary infection caused by a retrovirus, including forexample, human immunodeficiency virus (HIV). In addition, this inventionprovides methods of using pyrimidine derivatives as antitumor,antituberculosis, anti-Mycobacterium avium complex, antibiotic,antimalarial, antifungal and antiprotozoal agents and as synergisticagents with sulfonamides in such patients.

[0023] This invention also provides methods of using pyrimidinederivatives for therapeutic and/or prophylactic purposes as antitumoragents or to otherwise destroy or minimize growth or proliferation ofcancerous cells in cancer patients.

[0024] It is an object of this invention to provide pyrimidinederivative compounds, and pharmaceutically acceptable salts thereof, forsubstantially inhibiting dihydrofolate reductase enzymes.

[0025] It is an object of this invention to provide pyrimidinederivative compounds, and pharmaceutically acceptable salts thereof, forsubstantially inhibiting thymidylate synthase enzymes.

[0026] It is an object of the present invention to provide pyrimidinederivative compounds, and pharmaceutically acceptable salts thereof,having antitumor, antituberculosis, anti-Mycobacterium avium complex,antibiotic, antimalarial, antifungal or antiprotozoal activity includingsynergistic activity with sulfonamides and/or other agents.

[0027] It is a further object of this invention to provide pyrimidinederivative compounds, and pharmaceutically acceptable salts thereof,having effective activity against secondary infections, such as forexample infections caused by Pneumocystis carinii, Toxoplasmosis gondii,Mycobacterium tuberculosis and Mycobacterium avium complex that occur inimmunocompromised patients, such as patients with AIDS.

[0028] It is another object of this invention to provide pyrimidinederivative compounds, and pharmaceutically acceptable salts thereof,having effective activity against tumors and other cancerous cells, suchas those caused by cancer.

[0029] It is an object of this invention to provide a method ofsynthesizing various pyrimidine derivative compounds, andpharmaceutically acceptable salts thereof.

[0030] It is a further object of this invention to provide methods ofusing in a patient a therapeutically effective amount of pyrimidinederivative compounds, or pharmaceutically acceptable salts thereof.

[0031] It is a further object of this invention to provide methods ofusing in a patient a prophylactically effective amount of pyrimidinederivative compounds, or pharmaceutically acceptable salts thereof.

[0032] These and other objects of the invention will be more fullyunderstood from the drawing and the following description of theinvention and the claims appended hereto.

BRIEF DESCRIPTION OF THE FIGURES

[0033]FIG. 1 shows a schematic diagram of methods of preparing2,4-diamino-5-substituted-pyrrolo[2,3-d]pyrimidines.

[0034]FIG. 2 shows compounds 1-9 as synthesized by the methods shown inFIG. 1.

[0035]FIG. 3 shows a schematic diagram of the methods of preparingN-[4-[N-[2,4-diaminofuro[2,3-d]pyrimidin-5-yl]methyl]amino]benzoyl]-L-glutamicacid and the N-9 methyl analogue thereof.

[0036]FIG. 4 shows a schematic diagram of the methods of preparing of acompound having formula 8.

[0037]FIG. 5 shows a schematic diagram of the methods of preparingseveral compounds having formula 7.

[0038]FIG. 6 shows a schematic diagram of the methods of preparing twoof compounds having formula 4.

[0039]FIG. 7 shows a schematic diagram of the methods of preparing twoof compounds having formula 4.

[0040]FIG. 8 shows a schematic diagram of the methods of preparingcompounds having formula 9.

[0041]FIG. 9 shows of compounds having formula 9 as synthesized by themethods shown in FIG. 8.

[0042]FIG. 10 shows a schematic diagram of methods of preparingtricyclic[2,3-d]pyrimidines.

[0043]FIG. 11 shows a schematic diagram of the methods of preparingpyrido[3,2-d]pyrimidines.

[0044]FIG. 12 shows a schematic diagram of the methods of preparingpyrido[3,2-d]pyrimidines.

[0045]FIG. 13 shows a schematic diagram of the methods of preparingpyrido[2,3-d]pyrimidines.

[0046]FIG. 14 shows a schematic diagram of the methods of preparingvarious 2,4-diaminopyrido[2,3-d]pyrimidines.

[0047]FIG. 15 shows a schematic diagram of the methods of preparing2,4-diamino-6-substituted-benzylaminopyrido[2,3-d]pyrimidines.

[0048]FIG. 16 shows a schematic diagram of the methods of preparing2,4-diamino-6-(anilinomethyl)pyrido[2,3-d]pyrimidines.

[0049]FIG. 17 shows a schematic diagram of the methods of preparingpyrido[3,2-d]pyrimidines.

[0050]FIG. 18 shows a schematic diagram of the methods of preparingpyrido[3,2-d]pyrimidines.

[0051]FIG. 19 shows a schematic diagram of the methods of preparing thecompounds having formula 13.

[0052]FIG. 20 shows a schematic diagram of the methods of preparingcompounds having formula 13.

[0053]FIG. 21 shows the level of protection of FaDu cells from MTX(FIGS. 21A, 21B) and compound 113-161 (FIGS. 21C, 21D), determinedaccording to Example 32.

[0054]FIG. 22 shows the protection at 120 hours of FaDu cells from MTXand 113-161, determined according to Example 32.

[0055]FIG. 23 shows a schematic diagram of the methods of preparing acompound having Formula 14, as described in Example 35.

[0056]FIG. 24 shows a schematic diagram of the methods of preparingcompounds having Formula 15, as described in Example 37.

DETAILED DESCRIPTION OF THE INVENTION

[0057] As used herein, the term “patients” means members of the animalkingdom including but not limited to human beings.

[0058] The pyrimidine derivative compounds of the present invention, andpharmaceutically acceptable salts and prodrugs thereof, and methods ofpreparing and using these compounds provide for the therapeutic andprophylactic treatment of secondary infections caused by Pneumocystiscarinii, Toxoplasmosis gondii, Mycobacterium tuberculosis andMycobacterium avium complex in immunocompromised patients. Such patientscould have a primary infection caused by a retrovirus including but notlimited to human immunodeficiency virus (HIV). As will be appreciated byone skilled in the art, embodiments of the compounds, andpharmaceutically acceptable salts thereof, of the present inventionwhich contain benzoyl-L-glutamate groups will not be applicable to thesemethods. That is because Pneumocystis carinii and Toxoplasmosis gondiiare not generally known to take up enough of the benzoyl-L-glutamateforms of these compounds to be effective.

[0059] In addition, these compounds function as antitumor,antituberculosis and anti-Mycobacterium avium complex, antibiotic,antifungal, antimalarial and antiprotozoal agents, and as synergisticagents with other compounds.

[0060] The compounds of this invention also provide for the therapeutictreatment of tumors, or other cancerous cells, in cancer patients. Asused herein, the term “cancer” refers to any type of cancer including,but not limited to, leukemia, lung cancer, colon cancer, CNS cancer,melanoma, ovarian cancer, renal cancer, prostate cancer, and breastcancer. Prophylactic treatment of cancer is also contemplated by thepresent methods, in which the growth and/or spread of cancerous cells isminimized.

[0061] Many of the compounds disclosed in the present invention havemechanisms of action including but not limited to antifolates. Thepyrrolo(2,3-d]pyrimidine compounds, furo[2,3-d]-pyrimidine compounds,pyrrolo[3,2-d] and pyrrolo[3,4-d]pyrimidine compounds,thieno[2,3-d]pyrimidine compounds, cyclopentapyrimidine compounds,cyclopenta-[d]pyrimidine compounds, pyrido[2,3-d]pyrimidine compounds,and pyrido[3,2-d]pyrimidine compounds, and pharmaceutically acceptablesalts, formulations and prodrugs thereof, of this invention inhibitdihydrofolate reductase (DHFR) enzymes. The DHFR enzymes are needed fornormal cell growth because they reduce dihydrofolate totetrahydrofolate. Tetrahydrofolate is a precursor of5,10-methylenetetrahydrofolate, which is essential for DNA replicationand thus cell growth. The derivatives of the present invention inhibitdihydrofolate reductase and consequently inhibit DNA synthesis.Inhibition of DNA synthesis results in cell death.

[0062] In addition, the pyrimidine derivative compounds of the presentinvention, and pharmaceutically acceptable salts thereof, inhibitthymidylate synthase (TS). TS, along with DHFR, forms part of the systemresponsible for the synthesis of deoxythymidylate (dTMP) fromdeoxyuridylate (dUMP). Inhibition of TS deprives the cell of thymidine,which is an essential component of DNA.

[0063] As used herein, the term “pharmaceutically acceptable salts”include, salts of the present pyrimidine derivative compounds which aresuitable for use in pharmaceutical applications. One skilled in the artwould easily be able to determine whether a salt form of any givencompound is suitable for use as a pharmaceutical. Examples ofpharmaceutically acceptable salts include but are not limited to,acetate, formate, glucuronate, ethantate, sulfonate, or other saltsknown to those skilled in the art. “Pharmaceutically acceptableprodrugs” similarly refers to any prodrug formulations of the presentcompounds. A prodrug will be understood by those skilled in the art as achemical compound that is converted into an active curative agent byprocesses within the body. Other formulations comprising the pyrimidinederivatives described herein are also within the scope of the presentinvention.

[0064] In the pyrimidine derivative formulas presented herein, when Xand Y are either OH or NH₂, the enol form of the compounds isrepresented. The enol form is equivalent to and includes the keto formof the compounds.

[0065] As will be understood one skilled in the art, when any of thevariables used herein equal zero, that variable is not present in aparticular embodiment of the general formula. In any of the formulasdescribed herein, when any of the “A” variables equals zero, the “R”variable attached thereto also equals zero, and the “B” variable iseither zero or is attached directly to the carbon ring. When the “B”variable is zero, one or more of the “R” variables attached thereto canalso be zero or are attached directly to the A variable. When both the Aand B variables are zero, the R variables attached thereto can also bezero or are attached directly to the carbon ring. One skilled in the artwill understand this based upon the particular formulas included hereinand coupled with basic organic chemistry principles.

[0066] Some of the pyrrolo[2,3-d]pyrimidine compounds, andpharmaceutically acceptable salts thereof, of the present invention havethe general formula (1):

[0067] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0068] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0069] wherein Z₂ and Z₃ are different and are selected from the groupconsisting of R₄ and

[0070]  where Z₂ is R₄ when Z₃ is

[0071]  and Z₂ is

[0072]  when Z₃ is R₄;

[0073] wherein A is selected from the group consisting of CH and zero;

[0074] wherein B is selected from the group consisting of CH, nitrogen,N—CH₂, CH₂—N, CH₂—CH₂, oxygen, sulfur, sulfoxide, sulfone and zero;

[0075] wherein R₁ is selected from the group consisting of hydrogen, alower alkyl group, a nitroso group, a formyl group and zero and R₁ iszero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0076] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group and zero, and R₃ is zero when A is zero;

[0077] wherein R₄ is selected from the group consisting of hydrogen anda lower alkyl group;

[0078] wherein R₅ is selected from the group consisting of hydrogen anda lower alkyl group;

[0079] wherein R₈ is selected from the group consisting of naphthyl,mono-, di- and tri-substituted naphthyl, thionaphthyl, thiophenyl andhydroxyphenyl when R₁ is hydrogen and R₄ is hydrogen;

[0080] wherein R₈ is selected from the group consisting of phenyl,mono-, di- and tri-substituted phenyl, naphthyl, mono-, di- andtri-substituted naphthyl, pyridine and p-aroyl-L-glutamate when R₁ is alower alkyl group and R₄ is hydrogen;

[0081] wherein R₈ is selected from the group consisting of pyridine,phenyl, mono-, di- and tri-substituted phenyl, naphthyl, and mono-, di-and tri-substituted naphthyl and p-aroyl-L-glutamate when R₁ is zero;

[0082] wherein R₈ is selected from the group consisting of phenyl,mono-, di- and tri-substituted phenyl, naphthyl, mono-, di- andtri-substituted naphthyl and p-aroyl-L-glutamate when R₁ is hydrogen andR₄ is a lower alkyl group; and

[0083] wherein R₈ is not p-benzoyl-L-glutamate or pyridine when X is OH,A is zero, B is sulfur, R₄ is methyl and R₅ is hydrogen, and R₈ is notp-benzoyl-L-glutamate when X is OH, A is CH, B is CH, R₄ is hydrogen andR₅ is hydrogen; and

[0084] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from 1 to 6 carbons.

[0085] Preferred embodiments of formula 1 are further recited inTable 1. TABLE 1 L-M Compound X Y Bond A B R₁ R₃ R₄ R₅ R₈ 301 NH₂ NH₂dbl CH N H H H H 1-naphthyl 303 NH₂ NH₂ dbl CH N H H H H 4-OHphenyl 304NH₂ NH₂ dbl CH N CH₃ H H H 2,5- dimethoxy- phenyl 305 NH₂ NH₂ dbl CH NCH₃ H H H 3,4-dichloro- phenyl 306 NH₂ NH₂ dbl CH N CH₃ H H H 1-naphthyl307 NH₂ NH₂ dbl CH S —* H H H 3,4- dimethoxy- phenyl 308 NH₂ NH₂ dbl CHS — H H H 3,4-dichloro- phenyl 309 NH₂ NH₂ dbl CH S — H H H 1-naphthyl310 NH₂ NH₂ dbl CH S — H H H 2-naphthyl 312 NH₂ NH₂ dbl CH N CH₃ H H Hp-benzoyl-L- glutamate 313 OH NH₂ dbl CH N H H CH₃ H p-benzoyl-L-glutamate 314 OH NH₂ dbl CH N— H H CH₃ H p-benzoyl-L- CH₂ glutamate 315OH NH₂ dbl CH S — H CH₃ H 4-pyridine 317 OH NH₂ dbl — S — — CH₃ H3,4-dimethoxy- phenyl 318 OH NH₂ dbl — S — — CH₃ H 3,4-dichloro- phenyl319 OH NH₂ dbl — S — — CH₃ H 4-chloro- phenyl 320 OH NH₂ dbl — S — — CH₃H 4-NO₂phenyl 321 OH NH₂ dbl — S — — CH₃ H phenyl 322 OH NH₂ dbl — S — —CH₃ H 2-naphthyl

[0086] The most preferred embodiments of formula 1 are identified ascompounds 312 and 320.

[0087] The present invention is further directed to methods ofsynthesizing 5-substituted pyrrolo[2,3-d] pyrimidines. Synthesis ofthese compounds according to the methods of the present invention can beaccomplished by convergent synthesis. As will be understood by oneskilled in the art, convergent synthesis involves the production of anintermediate product from which numerous additional compounds can bemade. Here, the preferred intermediate product is 2,4-diamino-5-cyanopyrrolo-[2,3-d]pyrimidine.

[0088] More specifically, the present invention is directed to a methodof synthesizing a compound, and pharmaceutically acceptable saltsthereof, having the formula:

[0089] wherein R is selected from the group consisting of a lower alkylgroup, a p-aroyl-L-glutamate group, an aryl group, an allylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, anallyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, asubstituted alkoxyaryloxy group and a halogen; and

[0090] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons,comprising the steps of:

[0091] a) debrominating a pyrrole;

[0092] b) fusing the product of step a) with an amidine;

[0093] c) condensing the product of step b) with a nucleophile;

[0094] d) reducing the product of step c); and

[0095] e) purifying the compounds of step d).

[0096] Preferably, the debromination of step a) is performed in amixture of dimethylformamide (DMF) and methanol under hydrogenation inthe presence of a palladium catalyst. Either a 5% Pd—BaCO₃ or a 10% Pd—Ccatalyst can be used; 5% Pd—BaCO₃ is preferred. Typically, thedebromination step will be completed in about 3 hours, and the reactionperformed under hydrogen at a pressure of between about 40 and 60 psi,preferably at about 50 psi.

[0097] The fusion of step b) is preferably performed withchlorformamidine hydrochloride, and more preferably performed by heatinga uniformly stirred suspension of the product of step a) andchlorformamidine hydrochloride in a liquid heat transfer media andheating to a temperature of between about 150° C. and 180° C.,preferably between about 160° C. and 170° C., for a period of betweenabout 36 and 50 hours, preferably about 48 hours. Any liquid heattransfer media can be used, including Dowtherm-A®, available from DowChemical Company.

[0098] The selective nucleophile as disclosed in condensing step c) isselected from the group consisting of an aniline,diethyl(p-aminoaroyl)-L-glutamate, N-methyl anddiethyl(p-aminoaroyl)-L-glutamate. Any suitable aniline can be used,including but not limited to an aniline of the formula

[0099] wherein R₆ is selected from the group consisting of3′,4′,5′-trimethoxy, 3′,4′-dimethoxy, 4′-methoxy, 2′,5′-dimethoxy,2′,5′-diethoxy, 3′,4′-dichloro, 2′,3′-tetramethyl, hydrogen trimethoxy,dimethoxy and monomethoxy groups, trihalo, dihalo and monohalo groups,trialkyl, dialkyl and monoalkyl groups and combinations of methoxygroups, halo groups and lower alkyls.

[0100] Anilines of formula 3 are preferred for use in the methods of thepresent invention.

[0101] Preferably, the condensation of step c) is performed in 70% to80% acetic acid under hydrogenation, and in the presence of a Raneynickel catalyst. Hydrogenation times of from about 24 to 72 hours, andhydrogenation pressures of between about 50 and 60 psi, preferably 55psi, are preferred. Under these conditions, the Schiff bases areexpected to form.

[0102] Alternatively, condensation can be accomplished by heating amixture of the product of step b) with formaldehyde and Raney nickel ata temperature between about 70° and 90° C., preferably 80° C., for about2 hours.

[0103] Reduction of the Schiff bases, step d), is preferably performedby stirring a solution of the product of step c) in methanol at roomtemperature using NaCNBH as the reducing agent. In addition, 50%methanolic hydrochloric acid or glacial acetic acid can be used tomaintain the pH of the reaction mixture at about 2. The reduction stepshould take about 4 hours.

[0104] Preferably, the purification of step e) is performed by a methodselected from the group consisting of silica gel column chromatographyand dissolution of the product of step d) in methanol, filtration,evaporation of the filtrate, and trituration of the residue in anhydrousdiethylether.

[0105] When either diethyl(p-aminoaroyl)-L-glutamate or N-methyldiethyl(p-aminoaroyl)-L-glutamate are used in the condensation of stepc), an additional hydrolysis step, step f), is preferably performedfollowing step e). Preferably, the hydrolysis of step f) is accomplishedby stirring a solution of the product of step e) in a 1:1 sodiumhydroxide:methanol solution at room temperature for between about 60 and84 hours, preferably about 72 hours.

[0106] Specific embodiments of these methods are discussed in theexamples below.

[0107] Preferred embodiments of the compounds produced by the methods ofthe present invention are further recited in Table 2. TABLE 2 CompoundNumber R Group 1 3,4,5-trimethoxyphenyl 2 3,4-dimethoxyphenyl 32,5-dimethoxyphenyl 4 4-methoxyphenyl 5 2,5-diethoxyphenyl 63,4-dichlorophenyl 7 2′,3′-(CH)₄phenyl 8 phenyl 9 p-benzoyl-L-glutamate

[0108] Compounds 4, 8 and 9, as defined in the above table, arepreferred for therapeutically treating cancer patients, and compounds 4and 8 for therapeutically and prophylactically treating infectionscaused by Pneumocystis carinii and Toxoplasmosis gondii.

[0109] The present invention also provides pyrrolo[2,3-d]pyrimidinecompounds, and pharmaceutically acceptable salts thereof, having theformula (9):

[0110] wherein Q is selected from the group consisting of nitrogen andsulfur;

[0111] wherein R is selected from the group consisting of a lower alkylgroup, a p-aroyl-L-glutamate group, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, asubstituted alkoxyaryloxy group and a halogen;

[0112] wherein R₁₀ is selected from the group consisting of hydrogen anda lower alkyl group; and

[0113] wherein R₁₀ is a lower alkyl when Q is nitrogen.

[0114] Methods of synthesizing compounds having formula 9 as describedabove are also provided. These methods comprise the steps of:

[0115] a) debrominating a pyrrole;

[0116] b) fusing the product of step a) with an amide;

[0117] c) condensing the product of step b) with a nucleophile;

[0118] d) reducing the product of step c); and

[0119] e) purifying the compounds of step d); wherein the nucleophile ofstep c) is a compound having the general structure

[0120] wherein Q is selected from the group consisting of nitrogen andsulfur;

[0121] wherein R is selected from the group consisting of a lower alkylgroup, a p-aroyl-L-glutamate group, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, asubstituted alkoxyaryloxy group and a halogen;

[0122] wherein R₁₀ is selected from the group consisting of hydrogen anda lower alkyl group; and

[0123] wherein R₁₀ is a lower alkyl when Q is nitrogen.

[0124] The present invention is also directed to furo[2,3-d]pyrimidinecompounds, and pharmaceutically acceptable salts thereof, having thefollowing general formula:

[0125] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0126] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0127] wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and

[0128]  where Z is R₄ when Z₁ is

[0129]  and Z is

[0130]  when Z₁ is R₄;

[0131] wherein A is selected from the group consisting of CH and zero;

[0132] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, nitrogen, oxygen, CH, N—CH₂, CH₂—N, CH₂—CH₂, andzero;

[0133] wherein R₁ is selected from the group consisting of hydrogen, alower alkyl group, a nitroso group, a formyl group and zero and R₁ iszero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0134] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl, an alkoxy, analkoxyaryloxy group, a halogen and zero but R₂ is not3,4,5-trimethoxyphenyl, 3,4,5-trichlorophenyl, 3,4-dichlorophenyl,2,5-dimethoxyphenyl or a p-benzoyl-L-glutamate when R₁ is hydrogen andR₄ is hydrogen, and R₂ is not p-benzoyl-L-glutamate when R₁ is methyl;

[0135] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group, and zero and R₃ is zero when A is zero;

[0136] wherein R₄ is selected from the group consisting of hydrogen, alower alkyl group, S—R₇ and

[0137]  where R₇ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted allylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl, an alkoxy, analkoxyaryloxy group, a halogen and zero; and

[0138] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from 1 to 6 carbons.

[0139] In preferred embodiments of formula 4, X, Y, L, M and R₄ are asdescribed above, A is CH, R₃ is H, B is S or N, R₁ is H or zero, and R₂is selected from the group consisting of bicyclic and tricyclic ringsystems. Preferably, R₂ is an unsubstituted or a mono, di ortrisubstituted acridine, quinone, carbazole, phenanthrenes,dibenzofuran, anthracene, fluorene or fluorenone including but notlimited to preferred embodiments wherein R₂ is selected from the groupconsisting of 1-anthracene, 2-anthracene, 1-fluorene, 2-fluorene,2-(7-bromo)fluorene, 2-(9-hydroxy)fluorene, 1-fluoren-9-one,2-fluoren-9-one, 3-fluoren-9-one, 4-fluoren-9-one,2-(3-bromo)fluoren-9-one, 2-(7-bromo)fluoren-9-one, 2-fluoren-9-ol,1-anthraquinone, 2-anthraquinone, 1-(9,10-dihydro)anthracene,2-(9,10-dihydro)anthracene, 3-(9-ethyl)carbazole, and2-(3-methoxy)dibenzofuran.

[0140] Particularly preferred embodiments of formula 4 are recited belowin Table 3. For all of the embodiments described in Table 3, X is NH₂, Yis NH₂ and the bond between L and M is a double bond. TABLE 3 Compound AB R₁ R₂ R₃ R₄ 159 CH S — phenyl H H 160 CH S — 1-naphthyl H H 161 CH S —2-naphthyl H H 162 CH N H 1-naphthyl H H 163 CH N H 2-naphthyl H H 164CH O — 2-naphthyl H H 165 CH N H 2-phenoxyphenyl H H 166 CH N H4-phenoxyphenyl H H 167 CH N H 2-phenylphenyl H H 169 CH N H2′,5′-dichlorophenyl H H 171 CH N CH₃ 3′,4′-dichlorophenyl H H 172 CH NCH₃ 3′,4′,5′-trichlorophenyl H H 175 CH N H 3′-methoxyphenyl H H 177 CHH — — H S-R₇* 178 CH H — — H S-R₇ ^(†) 179 — — — phenyl — H 420 CH S —phenyl H H 421 CH S — 1-naphthyl H H 422 CH S — 2-naphthyl H H 423 CH NH 1-naphthyl H H 424 CH N H 2-naphthyl H H 425 CH O — 2-naphthyl H H 426CH N H 2-phenoxyphenyl H H 427 CH N H 4-phenoxyphenyl H H 428 CH N H2-phenylphenyl H H 429 CH N CH₃ 2-naphthyl H H 430 CH N H2,5-dichlorophenyl H H 431 CH N CH₃ 3,4-dichlorophenyl H H 432 CH N CH₃3,4,5-trichlorophenyl H H 433 CH N H 3-methoxyphenyl H H 434 CH N CH₃2,5-dimethoxyphenyl H H 111-157B CH N H 1-anthracene H H 111-178 CH N H2-anthracene H H 111-183 CH N H 1-fluorene H H 111-184 CH N H 2-fluoreneH H 111-190 CH N H 2-methoxy(dibenzofuran) H H 111-191 CH N H N-ethylcarbazole H H 111-192 CH N H hydroxy fluorene H H 111-194 CH N H1-fluoren-5-one H H 111-204 CH N H 1-fluoren-5-one H H

[0141] Compounds 161 and 167, as well as compounds 422 and 428, asdescribed in Table 3 are most preferred for therapeutically treatingcancer patients, and therapeutically and prophylactically treatinginfections caused by Pneumocystis carinii and Toxoplasmosis gondii.

[0142] The present invention is also directed to methods forsynthesizing the compound, and pharmaceutically acceptable saltsthereof, having the formula

[0143] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0144] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0145] wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and

[0146]  where Z is R₄ when Z₁ is

[0147]  and Z is

[0148]  when Z₁ is R₄;

[0149] wherein A is selected from the group consisting of CH and zero;

[0150] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, nitrogen, oxygen, CH, N—CH₂, CH₂—N, CH₂—CH₂, andzero;

[0151] wherein R₁ is selected from the group consisting of hydrogen, alower alkyl group, a nitroso group, a formyl group and zero and R₁ iszero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0152] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero, but R₂ is not3,4,5-trimethoxyphenyl, 3,4,5-trichlorophenyl, 3,4-dichlorophenyl,2,5-dimethoxyphenyl or p-benzoyl-L-glutamate when R₁ is hydrogen and R₄is hydrogen, and R₂ is not p-benzoyl-L-glutamate when R₁ is methyl;

[0153] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group, and zero and R₃ is zero when A is zero;

[0154] wherein R₄ is selected from the group consisting of hydrogen, alower alkyl group, S—R₇ and

[0155]  where R₇ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, anallyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower allyl, an alkoxy, analkoxyaryloxy group, a halogen and zero; and

[0156] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from 1 to 6 carbons,comprising the steps of:

[0157] a) stirring a pyrimidine and a substituted acetone in a solvent;

[0158] b) purifying the product of step a);

[0159] c) performing nucleophilic displacement of the chloride in theproduct of step b); and

[0160] d) purifying the product of step c).

[0161] In one embodiment of the methods described above, step a) isperformed by stirring one equivalent each of2,6-diamino-4-hydroxypyrimidine and 1,3-dichloroacetone in DMP at roomtemperature for a period of between about 12 and 36 hours, preferably 24hours.

[0162] Purification of the product of step a) can be accomplished by anymeans known in the art; column chromatography is preferred. The productresulting from the purification step b) is not generally stable for longdurations at room temperature. It is, therefore, preferred that thenucleophilic displacement of step c) be performed within about 1 hour ofcompletion of step b).

[0163] The nucleophilic displacement of step c) can be accomplished byany of various compounds including those selected from the groupconsisting of (p-aminoaroyl)-L-glutamic acid, diethylN-(p-methylaminoaroyl)glutamate, and a nucleophile. As used in referenceto the methods for synthesizing a compound, and pharmaceuticallyacceptable salts thereof, having formula (4), nucleophile includes, butis not limited to aniline, substituted anilines, phenols, thiophenolsand substituted phenols and thiophenols.

[0164] When step c) is performed with (p-aminoaroyl)-L-glutamic acid,the purification of step d) is preferably performed by precipitating theproduct of step c) by diluting said product with a suitable solvent,preferably water, and separating said product from unreacted startingmaterials and impurities by any means known in the art; cellulose columnchromatography is preferred. Acidification of the product is thenpreferred.

[0165] When the nucleophilic displacement of step c) is performed withdiethyl N-[p-methylamino(aroyl)]glutamate, the purification of step d)is preferably accomplished by stirring the product of step c) with 1 Nsodium hydroxide at room temperature for between about 12 to 36,preferably 24 hours, followed by acidification.

[0166] Alternatively, when conducting the nucleophilic displacement with(p-aminoaroyl)-L-glutamic acid, the desired product can also be obtainedby reductive methylation of the intermediate with a suitable aldehyde,preferably formaldehyde, and sodium cyanoborohydride at a pH ofapproximately 6 to 7. Purification is then accomplished by any meansknown in the art, preferably by wet cellulose column, and acidificationof the product performed.

[0167] When the nucleophilic displacement reactions are accomplishedwith an aniline, the product of step b) is preferably mixed withanhydrous dimethylsulfoxide and two equivalents of potassium carbonatefor approximately 60 to 84 hours, preferably 72 hours, at roomtemperature. Heating the reaction mixture to between about 35° and 45°C. for a period of between about 60 and 84 hours, preferably 72 hours,increases the yield of the desired product. The product is then isolatedfrom impurities and other unreacted starting materials by any meansknown in the art. Preferably, isolation is accomplished by adding excesswater to the reaction mixture and stirring at room temperature for aperiod of approximately 6 to 8 hours to separate the product;chromatographic purification is then performed.

[0168] The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the general formula

[0169] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0170] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0171] wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and

[0172]  where Z is R₄ when Z₁ is

[0173]  and Z is

[0174]  when Z₁ is R₄;

[0175] wherein A is selected from the group consisting of CH and zero;

[0176] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, nitrogen, oxygen, CH, N—CH₂, CH₂—N, CH₂—CH₂, andzero;

[0177] wherein R₁ is selected from the group consisting of hydrogen, alower alkyl group, a nitroso group, a formyl group and zero and R₁ iszero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0178] wherein R₂ is selected from the group consisting of a lower alkylgroup, an aryl group, p-aroyl-L-glutamate, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

[0179] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group and zero, and R₃ is zero when A is zero;

[0180] wherein R₄ is selected from the group consisting of hydrogen andlower alkyl group; and

[0181] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons.

[0182] The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the general formula:

[0183] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0184] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0185] wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and

[0186]  where Z is R₄ when Z₁ is

[0187]  and Z is

[0188]  when Z₁ is R₄;

[0189] wherein A is selected from the group consisting of CH and zero;

[0190] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, nitrogen, oxygen, CH, N—CH₂, CH₂—N, CH₂—CH₂, andzero;

[0191] wherein R₁ is selected from the group consisting of hydrogen, alower alkyl group, a nitroso group, a formyl group and zero, and R₁ iszero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0192] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

[0193] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group and zero, and R₃ is zero when A is zero;

[0194] wherein R₄ is selected from the group consisting of hydrogen andlower alkyl group;

[0195] wherein R₅ is selected from the group consisting of hydrogen andlower allyl group; and

[0196] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons.

[0197] In preferred embodiments of formula 6, Y and X are NH₂, the bondbetween L and M is a double bond, A is zero, B is CH, R₁ is hydrogen, R₂is selected from the group consisting of phenyl and 3,4-dichlorophenyl,R₃ is zero, R₄ is selected from the group consisting of hydrogen andlower alkyl and R₅ is selected from the group consisting of hydrogen andlower alkyl.

[0198] The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the general formula:

[0199] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0200] wherein A is selected from the group consisting of CH and zero;

[0201] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, nitrogen, oxygen, CH, N—CH₂, CH₂—N, CH₂—CH₂, andzero;

[0202] wherein R₁ is selected from the group consisting of hydrogen, alower alkyl group, a nitroso group, a formyl group and zero and R₁ iszero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0203] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero;

[0204] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group, and zero, and R₃ is zero when A is zero;

[0205] wherein R₄ is selected from the group consisting of hydrogen andlower alkyl group; and

[0206] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons.

[0207] In preferred embodiments of formula 7, X and Y are NH₂, A iszero, B is CH, R₁ is hydrogen, R₂ is selected from the group consistingof 3,4,5-trimethoxy-benzyl, 3,5-dimethoxybenzyl, 2,5-dimethoxybenzyl,3,4-dichlorobenzyl, 2,6-dichloro-benzyl, 2,4-dichlorobenzyl,2—CH₂-naphthyl, C₄H₄benzyl and 4-benzyl-L-glutamate, R₃ is zero, R ishydrogen, and R₅ is selected from the group consisting of hydrogen andmethyl. The most preferred embodiments of formula (7) are illustrated inFIG. 5.

[0208] The present invention is also directed to compounds, andpharmaceutically acceptable salts thereof, having the formula:

[0209] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0210] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0211] wherein Z₄, Z₅ and Z₆ are different and are selected from thegroup consisting of R₄, R₅ and

[0212] wherein A is selected from the group consisting of CH, sulfur andzero;

[0213] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, nitrogen, oxygen, CH, N—CH₂, CH₂—N, CH₂—CH₂, andzero;

[0214] wherein R₁ is selected from the group consisting of hydrogen, alower alkyl group, a nitroso group, a formyl group and zero, and R₁ iszero when B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0215] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkylgroup, an alkoxy, an alkoxyaryloxy group, a halogen and zero;

[0216] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group and zero, and R₃ is zero when A is zero;

[0217] wherein R₄ is selected from the group consisting of hydrogen anda lower alkyl group;

[0218] wherein R₅ is selected from the group consisting of hydrogen anda lower alkyl group;

[0219] where R₄ is the same or different than R₅;

[0220] wherein each of said R₄, R₅ and

[0221]  substituents is used once; and

[0222] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons.

[0223] In a preferred embodiment of formula 8, X and Y are NH₂, the bondbetween L and M is single, A is sulfur, B is carbon, R₁ is hydrogen, R₂is phenyl and R₃ is zero.

[0224] The present invention is also directed to tricyclic [3,2-d] and[2,3-d]pyrimidine compounds, and pharmaceutically acceptable saltsthereof, having the general formula:

[0225] wherein X and Y are the same or different and are selected fromthe group consisting of OH, NH₂, H and CH₃;

[0226] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0227] wherein R₁₁ is selected from the group consisting of a loweralkyl group, an aryl group, p-aroyl-L-glutamate, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy, analkoxyaryloxy group, a halogen and zero; and

[0228] Q₃ is selected from the group consisting of oxygen, NH, sulfurand CH₂.

[0229] In a preferred embodiment of formula 10, X and Y are NH₂, thebond between L and M is double, Q₃ is oxygen, and R is selected from thegroup consisting of phenyl, 3,4,5-trimethoxyphenyl, 3,5-dimethoxyphenyl,2,4-dichlorophenyl, 3,4-dichlorophenyl, 2,6dichlorophenyl andp-benzoyl-L-glutamate.

[0230] The present invention is also directed to a method forsynthesizing the tricyclic pyrimidine compounds generally represented byformula (10). These methods generally include the steps of condensing abiselectrophile derived from a piperidine with a pyrimidine, preferably2,4-diamino-6-hydroxy pyrimidine. Any compatible piperidine can be used;the piperidine chosen will depend on the final pyrimidine compounddesired.

[0231] In a preferred method, the piperidine is 4-piperidinehydrochloride; protection of the piperidine is effected with ditertiarybutyl-decarbonate in DMF at room temperature. Bromination of thisprotected piperidine is then performed in chloroform at room temperatureto yield a mixed product containing a bromopiperidine hydrobromidecompound and an N-Boc brominated compound. These compounds are thencondensed with a pyrimidine at room temperature for about 36 to 50hours, preferably 48 hours. The resulting product is then reacted withthe desired benzyl halide in anhydrous DMSO and anhydrous potassiumcarbonate for between about 48 and 72 hours at room temperature.Alternatively, the resulting product can be reacted with abenzoyl-L-glutamic acid diethyl ester instead of a benzyl halide.

[0232] The desired products of the above synthesis methods can beisolated using any purification means known in the art; chromatographicpurification is preferred. The isolation of the products may besimplified by adding an excess of water to the reaction mixture andstirring at room temperature for between about 1 and 3 hours, whichallows the products to separate.

[0233] The present invention is also directed to pyrido[2,3-d] and[3,2-d] pyrimidine compounds and pharmaceutically acceptable saltshaving the general formula:

[0234] wherein X and Y may be the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃;

[0235] wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and

[0236]  where Z is R₄ when Z₁ is

[0237]  and Z is

[0238]  when Z₁ is R₄;

[0239] wherein Q₁ and Q₂ are the same or different and are selected fromthe group consisting of CH and nitrogen;

[0240] wherein A is selected from the group consisting of nitrogen, CH,sulfur and zero;

[0241] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, CH, oxygen, nitrogen and zero, but B is not sulfur,sulfoxide, sulfone, oxygen or nitrogen when A is sulfur;

[0242] wherein R₁ is selected from the group consisting of hydrogen, anitroso group, an aldehyde, a lower alkyl group, a formyl group andzero, and R₁ is zero when B is zero, oxygen, sulfur, sulfoxide orsulfone;

[0243] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, anaklyltriaryl group, a substituted diaryl group and a substituted triarylgroup, and each substituent of the substituted aryl group, diaryl group,and triaryl group is the same or different and is selected from thegroup consisting of a lower alkyl group, an alkoxy, a substitutedalkoxyaryloxy group, a halogen and zero;

[0244] wherein R₃ is selected from the group consisting of H, a loweralkyl and zero, and R₃ is zero when A is zero;

[0245] wherein R₁₂ is selected from the group consisting of hydrogen andmethyl;

[0246] but R₂ is not 2,5-dimethoxyphenyl when X is NH₂, Y is NH₂, Q₁ isCH, Q₂ is N, Z₁ is H, B is CH, R₁ is H and R₁₂ is methyl; and

[0247] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons.

[0248] In one embodiment of formula 11, X and Y are both NH₂, Q₁ isnitrogen, Q₂ is CH, Z₁ is hydrogen, A is zero, B is nitrogen, R₁ ishydrogen, R₂ is selected from the group consisting of phenyl,2-methoxyphenyl, 4-methoxyphenyl, 4-chlorophenyl, 3,4dimethoxyphenyl,2,5-dimethoxyphenyl, 3,4,5-trimethoxyphenyl and 3,4-C₄H₄phenyl, R₃ iszero and R₁₂ is hydrogen.

[0249] In another embodiment of formula 11, X and Y are both NH₂, Q₁ isnitrogen, Q₂ is CH, Z₁ is hydrogen, A is zero, B is sulfur, R₁ is zero,R₂ is selected from the group consisting of 2-methoxyphenyl,4-methoxyphenyl and 3,4-dimethoxy-phenyl, R₃ is zero and R₁₂ ishydrogen.

[0250] In another embodiment of formula 11, X and Y are both NH₂, Q₁ isnitrogen, Q₂ is CH, Z₂ is hydrogen, A is zero, B is nitrogen, R₁ ismethyl, R₂ is selected from the group consisting of3,4,5-trimethoxyphenyl, 2,5-dimethoxyphenyl, 3,4-dimethoxyphenyl,3,4,5-trimethoxyphenyl, phenyl, 3,4-C₄H₄phenyl and 2,3-C₄H₄phenyl, R₃ iszero and R₁₂ is hydrogen.

[0251] In another embodiment of formula 11, X and Y are both NH₂, Q₁ isnitrogen, Q₂ is CH, Z₁ is hydrogen, A is zero, B is sulfone, R₁ is zero,R₂ is selected from the group consisting of 2-methoxyphenyl,4-methoxyphenyl, and 3,4-dimethoxy-phenyl, R₃ is zero and R₁₂ ishydrogen.

[0252] The present invention is also directed to pyrido[2,3-d] and [3,2dpyrimidine compounds and pharmaceutically acceptable salts having thegeneral formula:

[0253] wherein X and Y may be the same or different and are selectedfrom the group consisting of OH, NH₂, H and CH₃;

[0254] wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and

[0255]  where Z is R₄ when Z₁ is

[0256]  and Z is

[0257]  when Z₁ is R₄;

[0258] wherein Q₁ and Q₂ are the same or different and are selected fromthe group consisting of CH and nitrogen;

[0259] wherein A is selected from the group consisting of nitrogen, CH,sulfur and zero;

[0260] wherein B is selected from the group consisting of sulfur,sulfoxide, sulfone, CH, oxygen, nitrogen and zero, but B is not sulfur,sulfoxide, sulfone, oxygen or nitrogen when A is sulfur;

[0261] wherein R₁ is selected from the group consisting of hydrogen, anitroso group, an aldehyde, a lower alkyl group, a formyl group andzero, and R, is zero when B is zero, oxygen, sulfur, sulfoxide orsulfone;

[0262] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, anaklyltriaryl group, a substituted diaryl group and a substituted triarylgroup, and each substituent of the substituted aryl group, diaryl group,and triaryl group is the same or different and is selected from thegroup consisting of a lower alkyl group, an alkoxy, a substitutedalkoxyaryloxy group, a halogen and zero;

[0263] wherein R₃ is selected from the group consisting of H, a loweralkyl and zero, and R₃ is zero when A is zero;

[0264] wherein R₁₂ is selected from the group consisting of hydrogen andmethyl; and

[0265] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons.

[0266] The present invention is also directed to 5-substituted2,4-diaminopyrrolo[2,3-d]pyrimidines and2-amino-4-oxo-pyrrolo[2,3-d]pyrimidines, and pharmaceutically acceptablesalts thereof, having the general formula:

[0267] wherein X is selected from the group consisting of OH and NH₂;

[0268] wherein Z and Z₁ are different and are selected from the groupconsisting of R₄ and

[0269]  where Z is R₄ when Z₁ is

[0270]  and Z is

[0271]  group when Z₁ is R₄;

[0272] wherein A is selected from the group consisting of CH, sulfur,nitrogen and zero;

[0273] wherein B is selected from the group consisting of CH, NH, N—CH₂,CH₂—N, CH₂—CH₂, O, S, sulfoxide, sulfone and zero, but B is not sulfur,sulfoxide, sulfone, oxygen or nitrogen when A is sulfur;

[0274] wherein R₁ is selected from the group consisting of H, a loweralkyl group, a nitroso group, a formyl group and zero, and R₁ is zerowhen B is zero, oxygen, sulfur, sulfoxide or sulfone;

[0275] wherein R₂ is selected from the group consisting of a lower alkylgroup, p-aroyl-L-glutamate, an aryl group, an alkylaryl group, asubstituted aryl group, a substituted alkylaryl group, a diaryl group, atriaryl group, an alkyldiaryl group, an alicyclic hydrocarbon group, analkyltriaryl group, a substituted diaryl group, and a substitutedtriaryl group, :and each substituent of the substituted aryl group,diaryl group, triaryl group, or the substituted alkylaryl group,alkyldiaryl group, or alkyltriaryl group is the same or different and isselected from the group consisting of a lower alkyl group, an alkoxy,alkoxyaryloxy group, halogen and zero;

[0276] wherein R₃ is selected from the group consisting of hydrogen, alower alkyl group and zero, and R₃ is zero when A is zero;

[0277] wherein R₄ is selected from the group consisting of hydrogen, anda lower alkyl group; and

[0278] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1-6 to carbons.

[0279] Preferred embodiments of formula 13 are represented in thefollowing table: TABLE 4 Cmpd. X A B R₁ R₂ R₃ R₄ 402 NH₂ CH CH Hp-benzoyl-L-glutamate H H 403 NH₂ CH N H p-benzoyl-L-glutamate H H 404OH CH CH H p-benzoyl-L-glutamate H H 405 OH CH N H p-benzoyl-L-glutamateor H H substituted aryl rings 406 OH — S — p-benzoyl-L-glutamate — CH₃408 OH CH N H 2,5-dimethoxyphenyl H H 409 OH CH N H 3,5-dimethoxyphenylH H 410 OH CH N H 2,4-dimethoxyphenyl H H 411 OH CH N H3,4,5-trimethoxyphenyl H H 412 OH CH N H 2,5-dichlorophenyl H H 413 OHCH N H 3,5-dichlorophenyl H H 414 OH CH N H 2,4-dichlorophenyl H H 415OH CH N H 3-chlorophenyl H H

[0280] Particularly preferred embodiments are represented by compounds402 and 405. The substituted aryl rings of compound 405 can be anysubstituted aryl ring, including but not limited to an aryl group, analkylaryl group, a substituted aryl group, a substituted alkylarylgroup, a diaryl group, a triaryl group, an alkyldiaryl group, analicyclic hydrocarbon group, an alkyltriaryl group, a substituted diarylgroup, and a substituted triaryl group, and each substituent of thesubstituted aryl group, diaryl group, triaryl group, or the substitutedalkylaryl group, alkyldiaryl group, or alkyltriaryl group is the same ordifferent and is selected from the group consisting of a lower alkylgroup, an alkoxy, alkoxyaryloxy group, halogen and zero. Suitablesubstituents include but are not limited to substituted andunsubstituted acridine, anthracenes, fluorenes, fluorenones, carbazoles,dibenzofuran and phenanthrenes.

[0281] Compounds represented generally by formula 13, particularly thosedescribed above, and more particularly compound 404, have shownexcellent antitumor activity. It has been observed that a 2,4-diaminosubstituted pyrimidine ring can contribute to potent DHFR inhibitoryactivity. Compounds 402 and 403 have particularly strong DHFR inhibitorypotency. An unexpected result of the present invention, is the potentinhibitory activity of Compound 404 against both DHFR and TS.

[0282] Other pyrrolo[2,3-d]pyrimidines, and pharmaceutically acceptablesalts, formulations, and prodrugs thereof, encompassed by the presentinvention have the general formula:

[0283] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0284] wherein Z₄ and Z₅ are different and are selected from the groupconsisting of R₁₄ and

[0285]  where Z₄ is R₁₄ when Z₅ is

[0286]  and Z₄ is

[0287]  when Z₅ is R₁₄;

[0288] wherein A₁ is CH;

[0289] wherein B₁ is CH;

[0290] wherein R₁₃ is selected from the group consisting of H, a loweralkyl group, a substituted or unsubstituted aralkyl group, and asubstituted or unsubstituted aryl group;

[0291] wherein R₁₄ is selected from the group consisting of H and alower alkyl group;

[0292] wherein R₁₅ is selected from the group consisting of H and alower alkyl group;

[0293] wherein R₁₆ is selected from the group consisting of H and alower alkyl group;

[0294] wherein R₁₇ is selected from the group consisting of aryl,diaryl, triaryl, mono-, di- or tri-substituted aryl, mono-, di- ortri-substituted diaryl, mono-, di- or tri-substituted triaryl,substituted or unsubstituted heteroaryl, and p-aroyl-L-glutamate andeach of said substituents is independently selected from the groupconsisting of a substituted or unsubstituted lower alkyl group, asubstituted or unsubstituted alkoxy, a substituted or unsubstitutedalkoxyaryloxy group and a halogen;

[0295] wherein R₁₈ is a lower alkyl group; and

[0296] wherein each lower alkyl group is independently selected from thegroup consisting of lower alkyl groups having from about 1 to 6 carbons.

[0297] Several preferred embodiments of formula 14 are illustrated inTable 5 below. For all of these compounds, L and M are carbon and thebond between L and M is double, Z₅=R₁₄=H, R₁₅ is H, R₁₆ is H, and R₁₈ ismethyl. TABLE 5 PREFERRED EMBODIMENTS OF FORMULA 14 Compound R₁₃ R₁₇113-102 benzyl phenyl 113-103 H phenyl 113-118 benzyl 4-methoxy phenyl113-125 H 4-methoxy phenyl 113-143 benzyl 3-methoxy phenyl 113-143B H3-methoxy phenyl 113-148 benzyl 2-methoxy phenyl 113-154B H 2-methoxyphenyl 113-149 benzyl 3,4,5-trimethoxy phenyl 113-154A H3,4,5-trimethoxy phenyl 113-151 benzyl p-benzoyl-L-glutamate 113-161 Hp-benzoyl-L-glutamate

[0298] Particularly preferred embodiments are represented by compounds113-143, 113-149 and 113-154B.

[0299] The present invention is further directed tofuro[2,3-d]pyrimidine compounds, and pharmaceutically acceptable saltsand prodrugs thereof, having the general formula:

[0300] wherein L and M are selected from the group consisting of carbonand CH, the chemical bond between L and M is selected from the groupconsisting of a single bond and a double bond, L and M are carbon whenthe bond is a double bond, and L and M are CH when the bond is a singlebond;

[0301] wherein Z₆ and Z₇ are different and are selected from the groupconsisting of R₁₄ and

[0302]  where Z₆ is R₁₄ when Z₇ is

[0303]  and Z₆ is

[0304]  when Z₁ is R₁₄;

[0305] wherein A₂ is selected from the group consisting of carbon andCH;

[0306] wherein B₂ is selected from the group consisting of carbon andCH;

[0307] wherein the chemical bond between A₂ and B₂ is selected from thegroup consisting of a single bond and a double bond, A₂ and B₂ arecarbon when the bond is a double bond and A₂ and B₂ are CH when the bondis a single bond;

[0308] wherein R₁₄ is selected from the group consisting of H and alower alkyl group;

[0309] wherein R₁₅ is selected from the group consisting of H and alower alkyl group;

[0310] wherein R₁₆ is selected from the group consisting of H and alower alkyl group;

[0311] wherein R₁₇ is selected from the group consisting of aryl,diaryl, triaryl, mono-, di- or tri-substituted aryl, mono-, di- ortri-substituted diaryl, mono-, di- or tri-substituted triaryl,substituted or unsubstituted heteroaryl, and p-aroyl-L-glutamate andeach of said substituents is independently selected from the groupconsisting of a substituted or unsubstituted lower alkyl group, analkoxy, a substituted or unsubstituted alkoxyaryloxy group and ahalogen; and wherein each lower alkyl group is independently selectedfrom the group consisting of lower alkyl groups having from about 1 to 6carbons.

[0312] Preferred embodiments of formula 15 are illustrated in Table 6below. For all of these compounds, Z₇=R₁₄=H, R₁₅ is H, and R₁₆ ismethyl. TABLE 6 PREFERRED EMBODIMENTS OF FORMULA 15 COMPOUND L M A₂ B₂R₁₇ 120-74 carbon carbon CH CH p-benzoyl-L- glutamate 120-88 carboncarbon carbon carbon p-benzoyl-L- glutamate* 120-82 carbon carbon carboncarbon p-benzoyl-L- glutamate** 120-44 carbon carbon CH CH phenyl 120-35carbon carbon CH CH 3,4,5- trimethoxy phenyl 120-54 carbon carbon CH CH3,4-dimethoxy phenyl 120-76 carbon carbon CH CH 3-methoxy phenyl120-70-2 carbon carbon CH CH 2-methoxy phenyl 120-58 carbon carbon CH CH3,4-dichloro phenyl 120-61 carbon carbon CH CH 2,3,4-trichloro phenyl120-67 carbon carbon CH CH 2,5-dichloro phenyl 120-69 carbon carbon CHCH 2,4-dichloro phenyl 120-49 carbon carbon CH CH 6-methoxy naphthyl120-92 carbon carbon CH CH naphthyl 120-101 carbon carbon CH CH fluorene120-103 carbon carbon CH CH biphenyl 120-103-2 CH CH CH CH biphenyl120-105 carbon carbon CH CH 2,5-dimethoxy phenyl 120-106 carbon carbonCH CH 2-chlorophenyl 120-106-2 CH CH CH CH 2-chlorophenyl 120-107 carboncarbon CH CH 4-chlorophenyl 120-38 carbon carbon carbon carbon phenyl###120-25 carbon carbon carbon carbon 3,4,5- trimethoxy phenyl### 120-50carbon carbon carbon carbon 3,4-dimethoxy phenyl### 120-71 carbon carboncarbon carbon 3-methoxy phenyl### 120-70-1 carbon carbon carbon carbon2-methoxy phenyl* 120-56 carbon carbon carbon carbon 3,4-dichlorophenyl# 120-57-2 carbon carbon carbon carbon 2,3,4-trichloro phenyl120-57 carbon carbon carbon carbon 2,3,4-trichloro phenyl## 120-37-1carbon carbon carbon carbon 2,5-dichloro phenyl* 120-37-2 carbon carboncarbon carbon 2,5-dichloro phenyl*** 120-66 carbon carbon carbon carbon2,4-dichloro phenyl* 120-46 carbon carbon carbon carbon 6-methoxynaphthyl### 120-91 carbon carbon carbon carbon naphthyl### 120-20 carboncarbon carbon carbon naphthyl* 120-48 carbon carbon carbon carbonbiphenyl 120-40-1 carbon carbon carbon carbon 2,5-dimethoxy phenyl120-64 carbon carbon carbon carbon 2-chloro phenyl* 120-60 carbon carboncarbon carbon 3-chloro phenyl* 120-65 carbon carbon carbon carbon4-chloro phenyl## 120-94 carbon carbon carbon carbon 4-carbethoxy phenyl120-96 carbon carbon CH CH 4-carbethoxy phenyl

[0313] As used herein, the term “lower alkyl group” refers to an alkylgroup having between 1 and 6 carbons and can include straight, branchedor cyclic alkyl chains. The number of carbons in each lower alkyl groupin each of the present pyrimidine derivative formulas can vary. Forexample, in any given formula, R₁ could represent a lower alkyl grouphaving 1 carbon, R₂ could represent a lower alkyl group having 2carbons, R₃ could represent a lower alkyl group having 3 carbons, and R₄could represent a lower alkyl group having 4 carbons.

[0314] Thus, for all of the formulas described above, the lower alkylgroups are the same or different and are independently selected fromstraight chain, branched chain or cyclic (alicyclic hydrocarbon)arrangements having one to about six carbon atoms and can be substitutedor unsubstituted, such as methyl, ethyl, propyl, i-propyl, butyl,i-butyl, t-butyl, pentyl, hexyl, cyclopropyl, cyclobutyl, cyclohexyl,cyclopropylmethyl or cyclobutylmethyl groups. The carbon atoms of thesestraight chain, branched chain or cyclic arranged lower alkyl groups mayhave one or more substituents for the hydrogens attached to the carbonatoms. Such substituents are described below. It will be understood thatwhen the lower alkyl group contains one or more substituents three maybe more than six carbons in the lower alkyl group, but the main carbonchain will typically have six or less carbons.

[0315] “Aryl” groups will be understood as referring to compounds whosemolecules have a ring structure, such as the six carbon ring of benzeneor multiple rings which are either fused or unfused, such as condensedsix carbon rings of other aromatic derivatives. Suitable aryl groupstherefore include for example phenyl, biphenyl, benzyl, naphthyl,phenanthrene and anthracene groups. Thus, the term “aryl” includesdiaryl and triaryl groups which would have two and three rings,respectively, which are further described below. The aryl-groups can besubstituted or unsubstituted with any number of substituents includingbut not limited to a lower alkyl group, a substituted or unsubstitutedalkoxy, a substituted or unsubstituted alkoxyaryloxy group and ahalogen. Suitable substituted aryl groups include for example: mono-,di- and tri-substituted alkoxy phenyl groups; mono-, di- andtri-halogenated phenyl groups; mono-, di- and tri-substituted alkylphenyl groups; mono-, di- and tri-substituted alkoxy benzyl groups andmono-, di- and tri-substituted halogenated benzyl groups. Similarlysubstituted diaryl and triaryl groups are also suitable. Also includedwithin the term “aryl” are heterocycles, heteroaromatics or heteroaryls,which terms are used interchangeably herein and which will be understoodto those skilled in the art as closed ring structures having at leastone atom in the ring which is not carbon, such as oxygen, nitrogen orsulfur. Examples include but are not limited to pyrimidines, indoles,thiophenes, furans, benzofurans, pyridines, pyrroles, purines, and thelike. “Heteroaryls” as used herein also refers to such ring structuresthat are part of larger ring structures, such as two or three memberring systems, which may be fused or unfused, in which one of the ringsis as described above. It will be understood that this list is not meantto be exhaustive, and that any aryl group, as that term is commonlyunderstood in the art, is within the scope of the present invention.

[0316] The terms “alkylaryl”, “aryl alkyl” and “aralkyl” refer to groupshaving an alkyl moiety attached to an aryl ring such as a phenyl orbenzyl ring. The alkyl moiety is preferably a lower alkyl chain havingone to about seven carbon atoms. This alkyl moiety may also containoxygen, nitrogen or sulfur atoms, such as for example methoxy groups.The aryl moiety of the alkylaryl group is an unsubstituted,mono-substituted, di-substituted or tri-substituted aryl group, as thatterm is described above.

[0317] As used herein, the term aroyl, such as for example when usedwithin the term p-aroyl-L-glutamate, refers to heteroaroyl, benzoyl,napthoyl, thiophenoyl, furophenoyl, pyrroyl, and any other “aroyl” asthat term would be understood by one skilled in the art. “Aroyl” isgenerally defined in the art as an aromatic or heteroaromatic compoundhaving a carbonyl moiety.

[0318] The “R” groups described in the above formulas can generally besubstituted or unsubstituted. Each substituent on each group isindependently selected from the group consisting of a substituted orunsubstituted lower alkyl group having one to about six carbon atoms,a-substituted or unsubstituted alkoxy group, for example methoxy, asubstituted or unsubstituted aryl, diaryl or triaryl group, asubstituted or unsubstituted heteroaryl, a substituted or unsubstitutedalkoxyaryloxy group and a halogen, for example fluorene, chlorine orbromine.

[0319] Examples of other “R” substituent groups included in the formulasdescribed above can include but are not limited to those wherein: thealkylaryl group is selected from the group consisting of an alkylphenyland alkylbenzyl group; the allyldiaryl group is selected from the groupconsisting of alkylnaphthyl, alkylbenzothiophene, alkylindene,alkylbenzofuran, alkylindole and alkylaminoquinoline; the alkyltriarylgroup is an alkylanthracyl group; the substituted aryl, diaryl andtriaryl group is selected from the group consisting of a mono-, di- andtri-substituted allylphenyl and alkylbenzyl group, alkylnaphthyl,alkylbenzothiophene, alkylindole, alkylbenzofuran, alkylindene,alkylaminoquinoline, alkylanthracyl; each substituted alkyldiaryl andalkyltriaryl group is selected from the group consisting of a mono-, di-and tri-substituted alkylnaphthyl, alkylbenzothiophene, alkylindole,alkylbenzofuran, alkylindene, alkylaminoquinoline and alkylanthracylgroup; and each substituent is the same or different and is selectedfrom the group consisting of methyl, ethyl, n-propyl, i-propyl, n-butyl,i-butyl, t-butyl, branched n-pentyl, branched pentyl, n-hexyl, branchedhexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl,cyclopropylmethyl, cyclobutylmethyl, methoxy group, chlorine atom,bromine atom and fluorene atom.

[0320] Examples of substituted or unsubstituted diaryl or triaryl(bicyclic or tricyclic) ring systems suitable for use in the presentinvention include but are not limited to acridine; anthracenes includingbut not limited to 1-anthracene, 2-anthracene,1-(9,10-dihydro)anthracene, 2-(9,10-dihydro)anthracene; quinonesincluding but not limited to 1-anthraquinone, 2-anthraquinone, fluorenesincluding but not limited to 1-fluorene, 2-fluorene,2-(7-bromo)fluorene, 2-(9-hydroxy)fluorene, 1-fluoren-9-one; fluorenonesincluding but not limited to 2-fluoren-9-one, 3-fluoren-9-one,4-fluoren-9-one, 2-(3-bromo)fluoren-9-one, 2-(7-bromo)fluoren-9-one,2-fluoren-9-ol; carbazoles including but not limited to3-(9-ethyl)carbazole; dibenzofuran including but not limited to2-(3-methoxy)dibenzofuran; and phenanthrenes. These ring systems canthemselves contain any number of the additional “R” group substituentsdiscussed above. For example, a diaryl ring system (two fused arylrings) could itself have one or more aryl substituents, as could atriaryl ring system (three fused aryl rings). The tricyclic ringssystems are preferred; their configuration increases the attractionbetween the compound and the enzyme and helps in the binding of thecompound and enzyme.

[0321] In some embodiments of this invention, the compounds, andpharmaceutically acceptable salts thereof, having one of the formulasdescribed above wherein X and Y are each NH₂, R₁ is selected from thegroup consisting of H, CH₃ and CHO, CH₃CHO, and zero and R, R₂, and R₁₇are selected from the group consisting of 2,5-dimethoxyphenyl,2,3,4-trimethoxyphenyl, 2,4,6-trimethoxyphenyl, naphthyl,4-methoxynaphthyl, anthracyl and methoxy anthracyl, fluorene,benzothiophene, indene, benzofuran, indole, aminoquinoline,2,3-dichlorophenyl, 2,4-dichlorophenyl, 2,5-dichlorophenyl,2,6-dichlorophenyl, 3,4-dichlorophenyl and 3,5-dichlorophenyl. R₃ is CH₃or hydrogen, R₄ is either hydrogen, methyl, ethyl, propyl and butylgroup, cyclopropyl, cyclobutyl and cyclohexyl or zero, B is selectedfrom the group consisting of nitrogen, carbon, sulfur and oxygen, A isselected from the group consisting of nitrogen, carbon and sulfur.

[0322] In other embodiments of this invention, compounds andpharmaceutically acceptable salts thereof, are provided having any ofthe above formulas wherein X and Y are each NH₂. R₁ is selected from thegroup consisting of H, CH₃, NO and CHO, CH₃CHO, zero. R, R₂ and R₁₇ areselected from the group consisting of 2-methoxyphenyl, 3-methoxyphenyl,4-methoxyphenyl, 2,4-dimethoxyphenyl, 3,4-dimethoxyphenyl,3-5-dimethoxyphenyl, 2-chlorophenyl, 3-chlorophenyl, 4-chlorophenyl,2,3,4-trichlorophenyl, 2,4,5-trichlorophenyl, 2,4,6-trichlorophenyl,3,4,5-trichlorophenyl, 2-bromophenyl, 3-bromophenyl, 4-bromophenyl,2,4-dibromophenyl, 2,5-dibromophenyl, 2,6-dibromophenyl,3,4-dibromophenyl, 3,5-dibromophenyl, 2,4,6-tribromophenyl,2-fluorophenyl, 3-fluorophenyl, 4-fluorophenyl, 2,3-difluorophenyl,2,4-difluorophenyl, 2,5-difluorophenyl, 2,6-difluorophenyl,3,4difluorophenyl, 3,5-difluorophenyl, 2,3,4-trifluorophenyl,2,4,5-trifluorophenyl, 2,4,6-trifluorophenyl, 2-methylphenyl,3-methylphenyl, 4-methylphenyl, 2,3-dimethylphenyl, 2,4-dimethylphenyl,2,5-dimethylphenyl, 2,6-dimethylphenyl, -3,4-dimethylphenyl,3,5-dimethylphenyl, 2,4,5-trimethylphenyl, and 2,4,6-trimethylphenyl. R₃is CH₃ or hydrogen, and in formulas 3-5. R₄ is selected from the groupconsisting of hydrogen, methyl, ethyl, propyl, and butyl group,cyclopropyl, cyclobutyl, cyclohexyl and zero. B is selected from thegroup consisting of nitrogen, carbon, sulfur and oxygen and A isselected from the group consisting of nitrogen, carbon and sulfur.

[0323] In other embodiments of this invention, compounds andpharmaceutically acceptable salts thereof, are provided having any ofthe above formulas wherein X and Y are each NH₂. R₁ is selected from thegroup consisting of H, CH₃, NO and CHO, CH₃CHO, zero. R, R₂ and R₁₇ areselected from the group consisting of methyl, ethyl, n-propyl, n-butyl,cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, 2-methoxybenzyl,3,4-dimethoxybenzyl, 2,3-dimethoxybenzyl, 3,4-dimethoxybenzyl,2,3,4-trimethoxybenzyl, 3,4,5-trimethoxybenzyl, 2-chlorobenzyl,3,4-dichlorobenzyl, 2,3-dichlorobenzyl, 2,4-dichlorobenzyl,2,5-dichlorobenzyl, 2,6-dichlorobenzyl, 2,6-dichlorobenzyl,3,4-dichlorobenzyl, 3,5-dichlorobenzyl, 2-bromobenzyl,3,4-dibromobenzyl, 2-fluorobenzyl, 3,4-difluorobenzyl,2,4difluorobenzyl, 2,5-difluorobenzyl, 2,6-difluorobenzyl and3,4-difluorobenzyl. R₃ is CH₃ and hydrogen, and in formulas 3-5 R₄ isselected from the group consisting of a hydrogen, methyl, ethyl, propyl,butyl group, cyclopropyl, cyclobutyl, cyclohexyl; B is selected from thegroup consisting of nitrogen, carbon, sulfur and oxygen; and A isselected from the group consisting of nitrogen, carbon and sulfur.

[0324] The present invention further relates to methods of using theabove-described compounds, and pharmaceutically acceptable saltsthereof, in therapeutically and/or prophylactically treating a patientwith an illness. As used herein, the term “illness” refers to cancer,infection by Pneumocystis carinii, infection by Toxoplasmosis gondii,Mycobacterium tuberculosis and Mycobacterium avium complex, or othersecondary infections arising in immunocompromised patients, such asthose with AIDS, and infections caused by bacteria, tuberculosis,malaria, fungi or protozoa.

[0325] A method of therapeutically treating a patient for an illnesscomprises the steps of:

[0326] a) employing a compound, or pharmaceutically acceptable saltsthereof, having any of the above formulas 1, 2 or 4-15;

[0327] b) incorporating said compound in a suitable pharmaceuticalcarrier; and

[0328] c) administering a therapeutically effective amount of saidcompound incorporated in said carrier to a patient.

[0329] As used herein, the term “suitable pharmaceutical carrier” refersto any pharmaceutical carrier known in the art, absent compatibilityproblems with the compounds of formula 1, 2 or 4-15. Preferred carriersinclude physiologic saline and 5% dextrose.

[0330] As used herein, the term “therapeutically effective amount”refers to that amount of any of said compounds of formulas 1, 2 or 4-15incorporated in a suitable pharmaceutical carrier which is required tobring about a desired effect, such as reducing tumor size, destroyingcancerous cells or resisting/treating infection caused by organisms suchas Pneumocystis carinii, Toxoplasmosis gondii, Mycobacteriumtuberculosis and Mycobacterium avium complex.

[0331] As will be understood by one skilled in the art, atherapeutically effective amount of said compound can be administered byany means known in the art, including but not limited to, injection,parenterally, orally, or, where appropriate, topically.

[0332] It is well within the skill of one practicing in the art todetermine what dosage, and the frequency of this dosage, which willconstitute a therapeutically effective amount for each individualpatient, depending on the type of illness and the severity of suchillness. It is also within the skill of one practicing in the art toselect the most appropriate method of administering the compounds basedupon the needs of each patient.

[0333] Methods of prophylactically treating a patient for an illnesscomprise the steps of:

[0334] a) employing a compound, or pharmaceutically acceptable saltsthereof, having any of the formulas 1, 2 or 4-15 as described above;

[0335] b) incorporating said compound in a suitable pharmaceuticalcarrier; and

[0336] c) administering, a prophylactically effective amount of saidcompound incorporated in said carrier to a patient; wherein said illnessis selected from the group consisting of infection caused byPneumocystis carinii, Toxoplasmosis gondii, Mycobacterium tuberculosisand Mycobacterium avium complex.

[0337] As used herein, the term “prophylactically effective amount”refers to that amount of any of the compounds described above which willcause the body to generate antibodies in amounts sufficient to resistthe onset of infection caused by Pneumocystis carinii, Toxoplasmosisgondii, Mycobacterium tuberculosis or Mycobacterium avium complex inimmunocompromised patients.

[0338] As will be understood by one skilled in the art, aprophylactically effective amount of said compound can be administeredby any means known in the art, including but not limited to, injection,parenterally, orally,: or, where appropriate, topically.

[0339] It is well within the skill of one practicing in the art, todetermine what dosage, and the frequency of this dosage, which willconstitute a prophylactically effective amount for each individualpatient, depending on the type of illness, such as the type of cancer,and the severity of such illness. It is also within the skill of onepracticing in the art to select the most appropriate method ofadministering the compounds based upon the needs of each patient.

EXAMPLES

[0340] The following examples are set forth to illustrate variousembodiments of the invention, and should not be construed as limitingthe invention in any way. Standard test procedures familiar to thoseskilled in the art were used in the examples, such as those proceduresdescribed by Gangjee, A., et al., in “5-Arylthio-substituted2-amino4-oxo-6-methyl pyrrolo]2,3-d] pyrimidine antifolates asthymidylate synthase inhibitors and antitumor agents”, J. Med. Chem.,Vol. 38, pp. 4495-4502 (1995); “Effect of bridge region variation onantifolate and antitumor activity of classical 5-substituted2,4-diaminofuro[2,3-d]pyrimidines”, J. Med. Chem., Vol. 38, pp.3798-3805 (1995); and “Novel2,4-diamino-5-substituted-pyrrolo[2,3-d]pyrimidines As Classical andNon-Classical Antifolate Inhibitors of Dihydrofolate Reductases”, J.Med. Chem., Vol. 38, pp. 2158-2165 (Jun. 6, 1995) and referencesdisclosed therein.

Example 1

[0341] Compounds 1-6, 8, 301 and 303-312 were evaluated as inhibitors ofdihydrofolate reductase (DHFR) from Pneumocystis carinii (Pc),Toxoplasmosis gondii (Tg) and rat liver (RL). Performance of thesecompounds was compared with that of trimetrexate (TMQ), piritrexim(PTX), trimethoprim (TMP) and methotroxate (MTX), all of which arecurrently available. Trimetrexate is available from Warner-Lambert/ParkeDavis Pharmaceutical Research, Ann Arbor, Mich. Trimetrexate is approvedby the United States Food and Drug Administration as an approved newdrug for the treatment of Pneumocystis carinii infections in patientswith AIDS. PTX is an experimental anticancer agent in Phase II clinicaltrials and is also an agent against Pneumocystis carinii andToxoplasmosis gondii. TMP is an agent used against Pneumocystis cariniiinfection in conjunction with sulfonamides. MTX is a clinical usedanticancer agent.

[0342] The evaluations of Compounds 1-6, 8, 301 and 303-312 consisted ofdetermining the IC₅₀ values and selectivity ratios of each compoundagainst Pc DHFR, Tg DHFR and RL DHFR. The IC₅₀ value is theconcentration of a compound required to inhibit the dihydrofolatereductase activity by 50 percent (%). It will be understood by thoseskilled in the art that the lower the IC₅₀ value the more potent thecompound. The selectivity ratio is a measure of the selectivity of acompound for Pc DHFR or Tg DHFR and is expressed as the IC₅₀ value ofthe DHFR from rate liver (RL) divided by the IC₅₀ value of either the PcDHFR or the Tg DHFR, depending on which organism the compounds are beingtested against. For example, the selectivity ratio of a compound iscalculated by the following formula:$\frac{{IC}_{50}\quad {RL}\quad {DHFR}}{{IC}_{50}\left( {P_{c}\quad {DHFR}\quad {or}\quad T_{g}\quad {DHFR}} \right)}$

[0343] It will be understood by those skilled in the art that the higherthe selectivity ratio, the less toxic the compound is to mammaliandihydrofolate reductase, and thus, less toxic to the patient.

[0344] Table 7 sets forth the IC₅₀ values for Pc DHFR, RL DHFR and TgDHFR and the corresponding selectivity ratios for the compounds tested.TABLE 7 Inhibitory Concentrations (IC₅₀ μM) and Selectivity RatiosSelectivity Selectivity Ratio: Ratio: Compound # Pc DHFR¹ RL DHFR¹ RLDHFR/Pc DHFR Tg DHFR¹ RL DHFR/Tg  1 >23 56.3 <2.4 8.1 7.0  2 119.0 116.01.0 4.3 27.0  3 279.0 63.0 0.23 6.0 10.5  4 45.7 156.0 3.4 1.7 92.0 5 >21 70.0 <3.3 5.3 13.2  6 35.3 14.4 0.4 1.4 10.3  8 252.0 >252 >13.9 >65  9 0.038 0.044 1.20 0.21 0.21 301 307.0 59.3 0.2 1.1 53.9 30381.0 4.2 0.05 1.4 3.0 304 >12.0 >12.0 ND* 3.4 >4.0 305 28.90 3.0 0.111.0 3.0 306 209.0 8.20 0.04 0.87 9.43 307 11.10 16.7 1.50 2.60 6.42 30858.50 5.30 0.09 11.6 0.46 309 10.60 3.00 0.28 0.81 3.70 310 929.0 82.90.09 9.20 9.01 312 0.044 0.06 1.36 0.15 0.40 TMQ 0.042 0.003 0.07 0.010.30 PTX 0.038 0.001 0.04 0.01 0.14 TMP 12.0 133.0 11.1 2.7 49.0 MTX0.001 0.003 3.0 0.014 0.21

[0345] As can be seen from the above table, Compound 4 is almosttwo-times as selective at TMP and 306 times as selective as TMQ.Compound 8 similarly showed much higher selectivity than the compoundsknown in the art. These two compounds, therefore, represent preferredembodiments of the invention for the treatment of infections caused byPneumocystis carinii and Toxoplasmosis gondii as well as in treatment ofcancer patients as an antitumor agent or to destroy cancerous cells.With regard to Pneumocystis carinii, Compounds 4 and 8, with their highpotency and high selectivity, may be used clinically with a lesseramount of a leucovorin as compared to TMQ or may be used clinicallywithout the necessity of leucovorin, and thus greatly reduce the cost ofadministering these compounds to a patient.

Example 2

[0346] Compound 9 was tested for DHFR inhibition against the growth ofhuman leukemia CCRF-CEM cells. The performance of Compound 9 wasevaluated against MTX. Four different types of cells were used: CCRF-CEMcells were unaltered; R30dm cells had a decreased amount ofpolyglutamate synthase; R1 cells had a twenty-fold increase in wild typeDHFR protein and activity; and R2 cells were deficient in their abilityto uptake the compounds into the cell. Results are presented in Table 8below. TABLE 8 Growth Inhibition of Parental CCRF-CEM and Sublines withSingle, Defined Mechanisms of MTX Resistance During Continuous (0-120hours) Exposure to MTX and Compound 9 (EC₅₀ in nM) Compound CCRF-CEMR30dm R1 R2 MTX 14.5 ± 0.4 14.5 ± 0.5 595 ± 5  3100 ± 100 (n = 5) (n =2) (n = 2) (n = 2) 9 12.8 ± 2.2   36 ± 1   515 ± 25 1650 ± 200 (n = 5)(n = 2) (n = 2) (n = 2)

[0347] As can be seen from the results of the above table, Compound 9performed comparably to MTX. The example further demonstrates thatpolyglutamylation may play a limited role in the mechanism of action ofCompound 9, even in continuous exposure. Both the R₁ subline, withamplified DHFR expression, and the MTX-transport deficient R₂ sublinedisplayed resistance to MTX under continuous exposure.

Example 3

[0348] Compound 9 and MTX were also tested for growth inhibition of A253and FaDu human squamous carcinoma cell lines; neither of these celllines had any deficiencies. Results are presented below in Table 9.TABLE 9 Growth Inhibition of A253 and FaDu Human Squamous Carcinoma CellLines Following Continuous (120 hours) Exposure to MTX and Compound 9(EC₅₀ in nM) Compound A253 FaDu MTX 17 ± 1 31 ± 2 9 46 ± 4 22 ± 4

Example 4

[0349] Compound 9 and aminopterin were further tested for their activityas substrates for human leukemia cell CCRF-CEM folylpolyglutamatesynthetase. Aminopterin is a classical 2,4-diamino antifolate substrate.Results are presented below in Table 10. TABLE 10 Activity ofAminopterin and Compound 9 as Substrates for CCRF-CEM Human LeukemiaCell Folylpolyglutamate synthetase Substrate KM, μM V_(max, rel)V_(max)/K_(m(rel)) n Aminopterin 4.3 ± 0.2 1 0.23 ± 0.01 3 9 <1 0.72 ±0.07 >0.72 4

[0350] This example demonstrates that Compound 9 is a potent tumor cellgrowth inhibitor that shares determinates of response with MTX. It issimilar in potency to MTX as an inhibitor of the growth of humanleukemia and SCC cell lines in culture. DHFR is suggested as the targetof Compound 9, which is supported by the data showing relatively potentDHFR inhibition in vitro and the cross-resistance to Compound 9 of ahuman cell line having amplified expression of DHFR. Compound 9 may alsouse the MTX/reduced folate-transport protein for uptake as evidenced bythe cross-resistance of a human cell line in which this transport systemis defective. This example indicates that Compound 9 may be an excellentsubstrate for human FPGS with a very low K_(m). The slightcross-resistance to continuous exposure of the cell line having lowlevels of FPGS suggests that polyglutamate metabolites may play a rolein growth inhibition even under these conditions.

Example 5

[0351] Compound 9 was further tested for its in vitro anti-canceractivity by the National Cancer Institute. Results are presented inTable 11 below. GI₅₀ represents the concentration at which growth wasinhibited by 50%. TABLE 11 In Vitro Anti-Cancer Activity of Compound 9Panel/Cell Line GI₅₀ Leukemia CCRF-CEM ND HL-60 (TB) ND MOLT-4 NDRPMI-8226 ND SR >1.00E−04 Non-Small Cell Lung Cancer A549/ATCC <1.00E−08EKVX   3.97E−05 HOP-62 <1.00E−08 HOP-92   8.40E−06 NCI-H226 >1.00E−04NCI-H23   5.75E−08 NCI-H322M >1.00E−04 NCI-H522 >1.00E−04 Colon CancerCOLO 205 >1.00E−04 HCC-2998 <1.00E−08 HCT-116 <1.00E−08 HCT-15 <1.00E−08HT29 <1.00E−08 KM12   3.32E−05 SW-620   3.15E−06 CNS Cancer SF-268<1.00E−08 SF-295 <1.00E−08 SF-539 <1.00E−08 SNB-19 >1.00E−04 SNB-75<1.00E−08 U251 <1.00E−08 Melanoma LOX IMVI <1.00E−08 MALME-3M <1.00E−08M14 <1.00E−08 SK-MEL-2 >1.00E−04 SK-MEL-28 >1.00E−04 UACC-257 ND UACC-62<1.00E−08 Ovarian Cancer IGROV1 <1.00E−08 OVCAR-3 >1.00E−04OVCAR-4 >1.00E−04 OVCAR-5 >1.00E−04 OVCAR-8 <1.00E−08 SK-OV-3 >1.00E−04Renal Cancer 786-0 <1.00E−08 ACHN <1.00E−08 CAKI-1 <1.00E−08 RXF393 >1.00E−04 SN12C <1.00E−08 TK-10 >1.00E−04 UO-31 <1.00E−08 ProstateCancer PC-3 >1.00E−04 DU-145   1.09E−08 Breast Cancer MCF7 <1.00E−08MCF7/ADR-RES <1.00E−08 MDA-MB-231/ATCC >1.00E−04 HS 578T >1.00E−04MDA-MB-435   6.87E−07 MDA-N <1.00E−08 BT-549 >1.00E−04 T-47D >1.00E−04

[0352] As will be appreciated by one skilled in the art, the lower theGI₅₀, the more effective the compound. A GI₅₀ of less than 1.00×10⁻⁸indicates a very high potency, whereas a GI₅₀ greater than 1.00×10⁻⁴indicates that the compound was relatively inactive.

[0353] As demonstrated in Table 11 above, Compound 9 was shown to behighly selective against certain cell lines, with little or no activityagainst other cell lines. This result indicates that Compound 9 is not ageneral poison, but rather is very selective. For example, in the eightbreast cancer cell lines, Compound 9 was found to be highly potent, withGI₅₀ of less than 1.00×100⁻⁸ M, against the MCF7, MCF7-ADR-RES(resistant cell line) and the MDA—N cell lines, but was relativelyinactive, with GI₅₀ greater than 1.00×10⁻⁴ M against breast cancer celllines MDA-MB-231/ATCC, HS578T, BT-549 and T47D. The difference inactivity of these two sets of breast cancer cells is ten thousand fold,which indicates a very high degree of selectivity. This kind ofselectivity is repeated in all of the cell lines tested.

Example 6

[0354] The inhibitory concentration of Compounds 313 and 314 against TSwas determined. The performance of these compounds was measured againstPDDF, which is a standard TS inhibitor and experimental anticanceragent. The compounds were all tested against Lactobacillus casei andhuman lymphoma cells. Results are presented in Table 12 below: TABLE 12Inhibitory Concentrations (IC₅₀ in μM) Against TS Compound L. Casei TSHuman TS 313 180 180 314 360 ND PDDF 0.036 0.036

[0355] As can be seen from the above table, Compound 313 and 314, whentested, had surprisingly high inhibitory concentration when comparedwith that of PDDF.

Example 7

[0356] Compounds 315 and 317-322 were tested for their inhibitoryactivity against TS. The performance of these compounds was measuredagainst ZD1694, which is in Phase III clinical trials as an antitumoragent. All of the compounds were tested against human cells, L. casei,E. coli, and S. faecium. Results are presented in the Table 13 below. Ascan be seen in the table, compounds 318 and 320 are more potent thanZD1694. TABLE 13 Inhibitory Concentrations (IC₅₀ in μM) Against TSCompound Human L. Casei E. Coli S. Faecium ZD1694 0.22 8.8 5.3 8.8315 >25 >26 ND ND 317 2.4 >24 (33%) >24 (30%) >24 (12%) 318 0.13 4545 >45 (31%) 319 1.0 >26 (0%)  >26 (40%) 30 320 0.15 5.1 13 15 32130 >30 (0%)  >30 (30%) >30 (20%) 322 2.0 >25 (32%) >25 (36%) >25 (0%) 

Example 8

[0357] Compounds 317-320 were also tested for their ability to inhibitDHFR. Selectivity ratios were determined as measured againstPneumocystis carinii (c), Toxoplasmosis gondii (Tg) and rat liver (RL).Results are presented in Table 14 below. TABLE 14 Inhibition ofDihydrofolate Reductase (IC₅₀ in μM) Selectivity Selectivity Compound PcRL Ratio RL/Pc Tg Ratio RL/Tg 317 >21 >21 ND 20 >1 318 >20 >20 ND11.7 >1.7 319 40 24.6 0.62 3.1 7.94 320 >15 7470 ND 244 30.61

[0358] As can be seen from Table 14, Compound 320 is highly selectivefor Toxoplasmosis gondii DHFR.

Example 9

[0359] Compounds 317-322 were also tested for their ability to inhibitthe growth of FaDu human squamous cell carcinoma cell lines. Theperformance of these compounds was tested against PDDF, and AG331, whichis a TS inhibitor in Phase III clinical trials as an antitumor agent.Results are presented in Table 15 below. TABLE 15 Growth Inhibition ofthe FaDu Human Squamous Cell Carcinoma Cell Line By Continuous (120hours) Exposure to the Inhibitors Compound EC₅₀ μM n 317 >10 2 318insoluble — 319 6.7 ± 1.5 3 320 1.5 ± 0.4 3 321 >10 2 322 ND — PDDF 1.7± 0.2 4 AG331 1.0 ± 0.1 6

Example 10

[0360] The following example describes methods of synthesizing thecompounds represented by formula 2. These methods are illustrated inFIG. 1. Reference numerals and letters correspond with those of FIGS. 1and 2.

2-amino-3,4-dicyanopyrrole (11)

[0361] A mixture of about 4.0 grams (g) of Compound 10, about 4.0 g of5% Pd on BaCO₃, about 15 milliliters (ml) of DMF and about 25 mlmethanol was hydrogenated at about 50 psi for approximately 3 hours. Themixture was filtered through Celite®, a filtering compositioncommercially available from Johns-Manaville Products Corporation, andthe filtrate concentrated under reduced pressure to about 10 ml. About200 ml of cold water was added to the concentrate, and a light brownsolid was formed. This solid was collected by filtration to yield about1.60 g of Compound 11.

2,4diamino-5-cyanopyrrolo[2,3-d]pyrimidine (12)

[0362] A mixture of about 2.63 g of Compound 11 and about 2.5 g ofchlorformamidine hydrochloride in about 50 ml Dowtherm-A®, a liquid heattransfer media commercial available from Dow Chemical Company, washeated at between about 160° and 170° C. for approximately 48 hours,until Compound 11 could not be detected by thin layer chromatography(TLC). The mixture was cooled to room temperature, and about 50 ml ofEt₂O was added thereto. A greenish-brown solid resulted. The solid wasfiltered and washed with Et₂O to yield about 3.0 g of Compound 12.

2,4-diaminopyrrolo[2,3-d]pyrimidine-5-carboxaldehyde (13)

[0363] About 6.0 g of Raney Ni was added to a stirred solution of about2.0 g of Compound 12 and about 50 ml of HCOOH. The mixture was heated toabout 80° C. for about 2 hours, until no starting material could bedetected by TLC. The mixture was cooled to room temperature and filteredthrough Celite®. The filtrate was evaporated under reduced pressure,azeotroping with methanol to remove traces of HCOOH. The residue wasdissolved in about 25 ml of hot water, treated with Norit®, an activatedadsorption carbon commercially available from American Norit Company,Inc., and filtered through Celite®. The filtrate was neutralized withNH₄OH. The light brown precipitate which resulted was filtered and driedto yield about 1.40 g of Compound 13, which was immediately used insubsequent reactions without further purification.

2,4-diamino-5[[N-(3′,4′,5′-trimethoxyphenyl)imino]methyl]pyrrolo]2,3-d]pyrimidine(14a)

[0364] A solution containing about 1.30 g of Compound 12 and about 2.06g of 3,4,5-trimethoxyaniline in about 75 ml of 70% acetic acid andcontaining about 6.50 g of damp Raney Ni was hydrogenated at atmosphericpressure for about 24 hours at room temperature. The mixture was treatedwith Norit® and filtered through Celite® and the solvent removed fromthe filtrate by evaporation under reduced pressure. About 15 ml of coldwater was added to the residue, and the suspension was added to about100 ml of a stirred, cold, saturated solution of NaHCO₃. The mixture wasstirred for about 10 minutes and refrigerated for about 6 hours. Thebrown precipitate which formed was collected, washed with water, anddried. This product, containing Compound 14A, was washed repeatedly withEt₂O until no aniline was detected by TLC in the washings. The residuewas then dissolved in about 100 ml methanol and filtered, and thefiltrate evaporated under reduced pressure to near dryness. About 50 mlEt₂O was added to the solution, and the precipitate filtered to yieldabout 1.20 g of Compound 15a.

2,4-diamino-5-[(3′,4′,5′-trimethoxyanilino)methyl]pyrrolo[2,3-d]pyrimidine(1)

[0365] About 0.05 g NaCNBH₃ were added to a solution containing about0.20 g of Compound 15a in about 25 ml methanol. The pH was adjusted toabout 2 with a 50% methanol/hydrochloric acid solution. The mixture wascontinuously stirred at room temperature for about 4 hours. The solventwas evaporated to dryness, and cold water was added to the residue,which was neutralized with NH₄OH. The resulting precipitate wasfiltered, dried, and dissolved in a 9:1 mixture of CHCl₃/methanol. Thiswas applied to a silica gel column (2.4cm×20 cm) packed in CHCl₃. Thecolumn was eluted with a gradient of 1% methanol in CHCl₃ to 15%methanol in CHCl₃. Fractions corresponding to the product, as determinedby TLC, were pooled and evaporated to dryness under reduced pressure.The residue was triturated in cold Et₂O and filtered to yield about 0.10g of Compound 1.

2.4-diamino-5-[(3′,4′-dimethoxyanilino)methyl]pyrrolo[2,3-d]pyrimidine(2)

[0366] The Schiff base was prepared as described for Compound 15a,except that the reaction was carried out in 80% acetic acid and using3,4-dimethoxyaniline to yield about 1.20 g of Compound 15b. Reduction ofCompound 15b was carried out as described for Compound 1. The crudeproduct was dissolved in methanol and filtered. The filtrate wasevaporated under reduced pressure to dryness. The residue was trituratedin cold Et₂O and filtered to yield about 0.51 g of Compound 2.

2,4-diamino-5-[(4′-methoxyanilino)methyl]pyrrolo[2,3-d]pyrimidine (3)

[0367] The Schiff base was prepared as described above for Compound 15busing 4-methoxyaniline to yield about 0.32 g of Compound 15c. Reductionof Compound 15c was carried out as described for Compound 15b to yieldCompound 3.

2,4-diamino-5-[(2′,5′-dimethoxyanilino)methyl]pyrrolo[2,3-d]pyrimidine(4)

[0368] The Schiff base was prepared as described above for Compound 15busing 2,5-dimethoxyaniline to yield about 0.90 g of Compound 15d.Reduction of Compound 15d was carried out as described above forCompound 15b to yield about 0.25 g of Compound 4.

2,4-diamino-5-[[(2′,5′-diethoxyphenyl)iminolmethyl]-pyrrolo[2,3-d]pyrimidine(15e)

[0369] Method A, starting from Compound 13: A solution of about 1.15 gof Compound 13 and about 1.76 g of 2,5-diethoxyaniline (14e) in about 75ml 70% acetic acid containing about 5.75 g of damp Raney Ni washydrogenated at about 55 psi for about 12 hours at room temperature. Themixture was filtered through Celite® and the filtrate evaporated underreduced pressure. About 15 ml cold water were added to the residue andthis suspension was added to about 100 ml of a stirred, cold, saturatedsolution of NaHCO₃. The mixture was stirred for an additional 10 minutesand refrigerated for about 4 hours. The brown precipitate which resultedwas collected, washed with water, and dried. The crude productcontaining Compound 14e was washed repeatedly with Et₂O until no anilinecould be detected by TLC in the washings. The residue was then dissolvedin about 100 ml methanol and filtered, and the filtrate evaporated todryness under reduced pressure. About 20 ml Et₂O was added to thesolution, and the precipitate filtered to yield about 1.20 g of Compound15e.

[0370] Method B, starting from Compound 12: The Schiff base was preparedas described above for Compound 15b using 2,5-diethoxyaniline to yieldabout 0.90 g of Compound 15e, which was identical in all respects withthe sample prepared according to Method A described above.

2,4-diamino-5-[(2′,5′-diethoxyanilino)methyl]pyrrolo[2,3d]pyrimidine (5)

[0371] Reduction of Compound 15e was performed as described above forCompound 15b to yield about 0.36 g of Compound 5.

2,4-diamino-5-[(3′,4′dichloroanilino)methyl]pyrrolo[2,3-d]pyrimidine (6)

[0372] The Schiff base was prepared as described above for Compound 15busing 3,4-dichloroaniline to yield about 1.0 g of Compound 15f.Reduction of Compound 15f was performed as described above for Compound15b to yield about 0.34 g of Compound 6.

2,4-diamino-5-[(1′-naphthylamino)methyl]pyrrolo[2,3-d]pyrimidine (7)

[0373] The Schiff base was prepared as described above for Compound 15b,except that the reaction was carried out at about 30 psi for about 12hours at room temperature using 1-aminonaphthylene to yield about 0.92 gof Compound 15 g. Reduction of Compound 15 g was carried out asdescribed above for Compound 15b, except that glacial acetic acid wasused to adjust the pH to about 2. Crude product 7 was dissolved in a 9:1mixture of CHCl₃/methanol, which was loaded on a silica gel column(2.4cm×20 cm) packed in CHCl₃. The column was eluted with a gradient of1% methanol in CHCl₃ to 5% methanol in CHCl₃. Fractions corresponding tothe product, as determined by TLC, were pooled and evaporated underreduced pressure to dryness. The residue was triturated in cold Et₂O andthe suspension filtered to yield about 0.24 g of Compound 7.

2,4diamino-5-(anilinomethyl)pyrrolo[2,3-d]pyrimidine (8)

[0374]

[0375] The Schiff base was prepared as described above for Compound 15busing aniline to yield about 0.69 g of Compound 15h. Reduction ofCompound 15h was carried out as described for Compound 15b, except thatglacial acetic acid was used to adjust the pH to about 2. About 0.19 gof Compound 8 resulted.

N-[4-[N-](2,4-diaminopyrrolo[2,3-d]pyrimidin-5-yl)methyl]amino]benzoyl]-L-glutamicacid (9)

[0376] The Schiff base was prepared as described above for Compound 15busing diethyl(p-aminobenzoyl)-L-glutamate to yield about 1.39 g of thediethylester of Compound 16. Reduction of Compound 16 was carried out asdescribed above for Compound 15b to yield about 0.44 g of Compound 17.Hydrolysis of the esters was carried out by stirring a solution of about0.30 g Compound 17 in about 10 ml of 1N sodium hydroxide and 10 mlmethanol for about 72 hours at room temperature. The solvent wasevaporated to 5 ml, and the mixture was carefully acidified with glacialacetic acid in an ice bath. The tan precipitate which resulted wasfiltered, washed with water, and dried to yield about 0.23 g of Compound9.

Example 11

[0377] The DHFR inhibitory concentrations of Compounds 159-169, 171,172, 175, 177, 178, and 179 was determined. Performance of thesecompounds was measured against TMQ and TMP. Inhibitory concentration wasmeasured against Pneumocystis carinii and Toxoplasmosis gondii, as wellas rat liver. Results are presented in Table 16 below. TABLE 16Inhibitory Concentrations (IC₅₀, μM) and Selectivity Ratios of5-substituted furo[2,3-d]pyrimidines Compound Pc RL Selectivity TgSelectivity # DHFR DHFR Ratio RL/Pc DHFR Ratio RL/Tg 159 >26 252 ND >26ND 160 19 23 1.2 19 1.2 161 0.65 12.3 18.9 11.6 1.1 162 13.5 12 0.89 370.32 163 41 36.5 0.89 38 0.96 164 14 60.3 4.31 >42 ND 165 >12 >12 ND >12ND 166 8.1 16.2 2.00 32.4 0.50 167 7.7 187 17.79 45.4 3.02 169 50.9 71.91.4 >47 ND 171 44.8 >27 ND >27 ND 172 284 34.3 0.1 21.5 1.6175 >31.3 >31.3 ND >31.3 ND 177 8.6 >83 >10 >83 ND 178 >12 >12 ND >12 ND179 >27.9 >27.9 ND >27.9 ND TMQ 0.042 >0.003 0.07 0.01 0.30 TMP 12 13311.1 2.7 49.0

[0378] As can be seen from the above table, Compound 161 isapproximately 18 times more active than TMP and 1.7 times moreselective; Compound 161 is 271 times more selective than TMQ and only 15times less potent. These compounds, therefore, exhibit high selectivityfor Pc DHFR. Compound 167 was also more selective than the compoundsknown in the art.

Example 12

[0379] The following example provides methods for synthesizing compoundsof formula 4. Reference numerals correspond with those in FIGS. 6 and 7.

Methyl 4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)-ethanyl]benzoate (27)

[0380] About 2.64 g of tributylphosphine was added to a solutioncontaining about 0.79 g of2,4-diamino-5(chloromethyl)furo[2,3-d]pyrimidine (25) in about 10 mlanhydrous DMSO. The mixture was stirred at about 60° C. for about 2hours under nitrogen to form the phosphonium salt. The solution wascooled to room temperature, at which time 0.35 g of sodium hydride (60%dispersion in mineral oil) followed by about 0.72 g of methyl4-formylbenzoate (26) was added. The mixture was stirred for about 24hours at room temperature. The DMSO was removed by vacuum distillation.About 50 ml of ethylether was added to the residue and the supernatantdecanted after 10 minutes. About 50 ml of ethylether was added again,the residue stirred for about 1 hour, and the supernatant decanted. Thisprocess was repeated 3 more times; the mixture was then stored at 0° C.with about 50 ml ethylether. After about 18 hours, the mixture wasultrasonicated for 2 hours and cooled to 0° C. for a period of about 10hours. The resulting solid was filtered, washed with ethylether, airdried, washed with water and air dried again. The solid was thensuspended in about 250 ml of hot methanol. About 3 g of silica gel wereadded to the filtrate, and the suspension was evaporated to drynessunder reduced pressure. The silica gel plug was loaded on a dry silicagel column (2.4×20 cm) and flushed initially with CHCl₃ (500 ml). Thecolumn was then eluted sequentially with 100 ml portions of 1% to 8%methanol in CHCl₃. Fractions which showed a major spot at R_(f) 0.66, asdetermined by TLC, were pooled and evaporated to dryness. The resultingresidue was dissolved in glacial acetic acid and evaporated to dryness.This residue was redissolved in hot methanol and the solution stored at0° C. for about 72 hours. The resulting solid was filtered, washed withether, and dried to yield about 0.52 g of Compound 27.

Methyl 4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoate (28)and (±)-methyl4-]2-(2,4-diamino-5,6-dihydrofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoate(29)

[0381] About 0.31 g of 5% palladium on carbon was added to a solutioncontaining about 0.155 g of Compound 27 in about 20 ml of a 1:1 mixtureof methanol/DMF. The suspension was hydrogenated in a Parr apparatus atroom temperature and 25 psi of hydrogen pressure for 30 minutes. Thereaction mixture was filtered through Celite®, and the catalyst waswashed with about 30 ml of a 1:1 methanol/DMF mixture. The filtrate wasevaporated to dryness under reduced pressure, and the residue dissolvedin about 100 ml methanol. About 500 mg of silica gel was added to thesolution, and evaporated to dryness. The silica gel plug was loaded on adry silica gel column (2.4×16cm) and flushed with about 500 ml CHCl₃.The column was then eluted with a gradient of 1-9% methanol in CHCl₃,collecting 15 ml fractions. Fractions showing a single spot at R_(f)0.63, as determined by TLC, were pooled and evaporated to dryness, andthe residue was stirred in ether, filtered, and dried to yield about0.08 g of Compound 28. Later fractions from the column described above,showing a single spot at R_(f) 0.52, were pooled and evaporated todryness under reduced pressure, and the residue obtained was stirred inether, filtered, and dried to -yield about 0.02 g of Compound 29.

4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzoic acid (30)

[0382] About 1.5 ml of 1N sodium hydroxide was added to a solutioncontaining about 0.065 g of Compound 28 in about 10 ml of a mixture of2:1 methanol/DMSO. The mixture was stirred at room temperature for 18hours and evaporated to dryness under reduced pressure (oil pump). Theresidue was dissolved in about 5 ml water and 1 N HCl was added dropwiseto bring the pH of the solution to 5.5. The suspension was cooled to 5°C. for about 12 hours and filtered. The residue was washed sequentiallywith water, acetone and ether and dried to yield about 0.05 g ofCompound 30.

N-[4-[2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]-benzoyl]-L-glutamicacid (33)

[0383] About 45 microliters of triethylamine was added to a suspensioncontaining about 0.047 g of Compound 30 in about 3 ml anhydrous DMF, andthe mixture stirred under nitrogen at room temperature for about 5minutes. The solution was cooled to 0° C., about 42 microliters ofisobutylchloroformate was added, and the mixture stirred at 0° C. for 30minutes. About 0.077 g of diethyl-L-glutamate hydrochloride was added tothe reaction mixture, followed immediately by about 45 microliterstriethylamine. The mixture was slowly allowed to warm to roomtemperature, and stirred under nitrogen for a period of about 18 hours.The reaction mixture was then subjected to another cycle of activationusing ½ of the quantities listed above. The reaction mixture was warmedto room temperature and stirred for 24 hours and evaporated to drynessunder reduced pressure. The residue was dissolved in a 4:1 mixture ofCHCl₃/methanol and chromatographed on a silica gel column (2.4×15 cm),packed with CHCl₃/methanol (24:1), eluting with 24:1 CHCl₃/methanol.Fractions showing a single spot were pooled and evaporated to dryness.The residue was stirred in cold anhydrous ether and filtered to obtainabout 0.054 g of Compound 31. About 1 ml of 1N sodium hydroxide wasadded to a solution containing about 0.052 g of Compound 31 in about 5ml methanol and the solution stirred at room temperature for 24 hours.The methanol was evaporated under reduced pressure, the residuedissolved in about 5 ml water, and stirring was continued for anadditional 24 hours. The pH of the solution was then adjusted to 4.0 bydropwise addition of 1N HCl. The resulting suspension was stored at 5°C. for about 12 hours and filtered; the residue was washed well withwater and acetone and dried to yield about 0.044 g of Compound 33.

4-[[N-(tert-butyloxycarbonyl)amino]methyl]benzoic acid (39)

[0384] About 10 ml of 1N sodium hydroxide was added to a solutioncontaining about 1.5 1 g of 4-(aminomethyl)benzoic acid (32) in about 20ml of 1:1 dioxane/water. The mixture was stirred at room temperature forabout 12 hours, and evaporated to half of its original volume underreduced pressure. The pH of the solution was adjusted to 3 by dropwiseaddition of 50% aqueous HCl, while maintaining the temperature below 10°C. with an ice bath. The resulting suspension was diluted with water (70ml) and extracted with ethylacetate (3×50 ml). The combined organiclayers were washed with about 50 ml saturated NaCl, dried MgSO₄, andfiltered. The filtrate was evaporated to dryness under reduced pressureand the residue recrystallized from a mixture of ethylacetate/hexanes toyield about 2.10 g of Compound 39.

Diethyl-N-[4-[[N-(tert-butyloxycarbonyl)amino]methyl]benzoyl]-L-glutamate(34)

[0385] A solution containing about 1.26 g of Compound 39 in about 20 mlanhydrous DMF under nitrogen was cooled in an ice-salt bath. About 0.55ml of N-methylmorpholin was added to the cooled solution, followed 5minutes later by about 0.65 ml isobutylchloroformate. After stirring fora period of about 20 minutes, 1.20 g diethyl-L-glutamate hydrochloridewas added, followed immediately by about 0.55 ml N-methylmorpholin. Thereaction mixture was warmed to room temperature and stirred for 12hours. At this time, the activation cycle was repeated using ½ theamounts of reagents indicated above, after which the reaction mixturewas warmed to room temperature and stirred for an additional 12 hours.The solvents were removed under reduced pressure, and the residue wasdissolved in about 100 ml CH₂Cl₂, washed with about 75 ml water, 50 ml0.1 NHCl, and about 50 ml saturated NaCl. The organic layers were dried(MgSO₄) and filtered. The filtrate was evaporated under reduced pressureand the residue was flash chromatographed on silica gel (2.4×24 cm),eluting first with CH₂Cl₂ and then with 1% methanol in CH₂Cl₂. Fractionsshowing a single spot corresponding to the product were pooled andevaporated under reduced pressure to yield about 1.29 g of Compound 34.

Diethyl N-[4-(aminomethyl)benzoyl]-L-glutamate (35)

[0386] About 1.8 ml trifluoroacetic acid was added dropwise to a stirredsolution containing about 1.0 g of Compound 34 in about 20 ml CH₂Cl₂.The mixture was stirred at room temperature for 15 minutes, evaporatedto dryness under reduced pressure, and co-evaporated twice with about 30ml absolute ethanol. The residue was then subjected to columnchromatography on silica gel (1.5×15 cm), eluting with a gradient of5-10% methanol in CHCl₃ to yield about 0.68 g of Compound 35.

DiethylN-[4-[N-](2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl(amino)methyl(benzoyl)-L-glutamate(37)

[0387] About 0.28 g of anhydrous K₂CO₃ and about 0.67 g of Compound 35were added to a solution containing about 0.2 g of Compound 35 in about3 ml anhydrous DMSO. The reaction mixture was stirred under nitrogen atroom temperature for 24 hours. The temperature was then raised to about45° C. and the reaction continued for an additional 48 hours. Thereaction mixture was then cooled to room temperature, diluted with about50 ml water and stirred for about 8 hours. The solid that separated wasfiltered, washed with water, air dried, and dissolved in methanol. About1 g of silica gel was added to the solution and the suspensionevaporated to dryness under reduced pressure. The silica plug was loadedon a dry silica gel column (2.4×17 cm) and eluted with a gradient of1-7% methanol in CHCl₃. Fractions corresponding to the product werepooled and evaporated to dryness. The residue was triturated with coldanhydrous ether to yield about 0.25 g of Compound 37.

N-[4-[[N-](2,4-diaminofuro[2,3-d]pyrimidin-5-yl)methyl]amino]methyl]benzoyl]-L-glutamicacid (34)

[0388] About 1 ml of 1N sodium hydroxide was added to a solutioncontaining about 01 g of Compound 37 in about 10 ml of 2:1 methanol/THF.The mixture was stirred at room temperature for about 24 hours. Thevolatiles were removed under reduced pressure, and the residue dissolvedin about 5 ml water and stirred for an additional 24 hours. The solutionwas cooled in an ice bath and the pH adjusted carefully to 4.0 bydropwise addition of 1N HCl. The precipitate was collected byfiltration, washed well with water and ether, and immediately driedunder high vacuum to yield about 0.08 g of Compound 34.

Example 13

[0389] Compounds 33 and 34 were evaluated as inhibitors of Lactobacilluscasei DHFR, human recombinant (REC) DHFR and DHFR isolated from humanCCRF-CEM leukemic cells. The performance of these compounds was comparedwith that of MTX. The compounds were also evaluated as inhibitors of L.casei TS and human recombinant TS. The results are presented in Table 17below. TABLE 17 Inhibitory Concentrations (IC₅₀ in μM) Human L. REC L.casei CCRF-CEM Human REC casei Compound DHFR DHFR DHFR TS TS 33 1.0 0.10.25 220 200 34 >200 >200 30.5 63.0 >200 MTX 0.004 0.006 0.0007 170 ND

Example 14

[0390] Compounds 33 and 34 were also evaluated for their substrateactivity in CCRF-CEM human leukemia folylpolyglutamate synthetase; thecompounds were compared against aminopterin. Results are presented inTable 18 below. TABLE 18 Substrate Activity of Compounds 33 and 34 forCCRF-CEM Human Leukemia Cell Folylpolyglutamate Synthetase SubstrateK_(m), μM V_(max(rel)) V_(max)/K_(m(rel)) Aminopterin 4.8 ± 0.7 1 0.21 ±0.04 (N = 6) (N = 6) (N = 6) 33 8.5 ± 2.1 0.65 ± 0.01 0.07 ± 0.02 (N =3) (N = 3) (N = 3) 34 ND 0.6 ND (N = 5)

Example 15

[0391] Compounds 33 and 34 were also tested for their growth inhibitionof human T-lymphoblastic leukemia cell line CCRF-CEM, its MTX-resistancesubline (R₃₀dm), and human squamous cell carcinoma cell lines (FaDu andA235). The performance of these compounds was evaluated against MTX.Results are presented in Table 19 below. TABLE 19 Growth Inhibition(EC₅₀, μM) During Continuous Exposure (0 to 120 hours) Compound CCRF-CEMR30DM FaDu A253 33  0.29 ±  4.25 ± 0.018 ± 0.02 0.54 ± 0.09 0.01 0.05 (N= 2) (N = 3) (N = 3) (N = 2) 34  48.0 ± ND ND ND 23.0 (N = 2) MTX 0.014± 0.018 ± 0.017 ± 0.002 0.013 ± 0.0008 0.001 0.003 (N = 5) (N = 3) (N =10) (N = 5)

Example 16

[0392] The following example provides methods for preparing thepyrido[2,3-d]pyrimidine compounds and pharmaceutically acceptable saltsthereof as described above. Reference letters and numerals correspondwith those in FIG. 13. Guanidine (FIG. 13b) is condensed with2-amino-3,5-dicarbonitrile-4-R₁₃-pyridine (FIG. 13a), wherein R₁₃ is alower alkyl group having one to about seven carbon atoms as describedherein, in refluxing ethyl alcohol to produce2,4-diaminopyrido[2,3-d]pyrimidine-5-R₃-6-carbonitrile (FIG. 13c). Thisproduct, 2,4-diaminopyrido[2,3-d]pyrimidine-5-R₃-6-carbonitrile (FIG.13c), is then subjected to reductive condensation with an alkyl amine, asubstituted aniline or benzylamine derivative containing the R₂ group asdescribed herein, such as for example, 3,4,5-trimethoxyaniline, andRaney nickel in aqueous acetic acid solution, and preferably about 70%acetic acid solution, to form2,4-diamino-5-R₁₃-6-[[(R₂)amino]methyl]pyrido[2,3-d]pyrimidine (FIG.13d). The starting material 2-amino-3,5-dicarbonitrile-4-R₃-pyrimidine(FIG. 13a) may be synthesized by those skilled in the art by modifyingthe method of Piper, et al., J. Med. Chem., Vol. 29, p. 1080 (1986).

[0393] These methods further include adding the product represented inFIG. 13d to about 37% formaldehyde in acetonitrile at about 25° C.,adding sodium cyanoborohydride, glacial acetic acid and methanol, andrefrigerating the reaction mixture overnight to form2,4-diamino-5-R₁₃-6[[(R₂)methylamino]methyl]pyrido[2,3-d]pyrimidine(FIG. 13e).

[0394]2,4-Diamino-5-R₃-6[[(R₂)formylamino]methyl]pyrido[2,3-d]pyrimidine (FIG.13f) is prepared by reacting the product of FIG. 13d in about 98% formicacid as a solvent and acetic anhydride as a catalyst, removing thesolvent under reduced pressure, diluting the reaction product withmethanol and refrigerating the diluted reaction product overnight.

[0395]2,4-Diamino-5-R₃-6[[(R₂)nitrosoamino]methyl]pyrido[2,3-d]pyrimidine(FIG. 13g) is prepared by reacting a chilled solution of the product ofFIG. 13d in aqueous acetic acid and dimethyl formamide (DMF) and thenadding NaNO₂ (sodium nitrate) in water. This mixture is stirred at about0° C. to 5° C. for about two hours and then poured into dilute-sodiumhydroxide.

[0396] It will be appreciated by those skilled in the art that byfollowing the hereinbefore described methods of preparing the productsof FIGS. 13d, 13 e, 13 f and 13 g of this invention that the derivativesof the products of FIGS. 13d, 13 e, 13 f and 13 g can be similarlyprepared using the appropriate alkylamine, substituted aniline orbenzylamine derivative containing the R₂ group as described herein.

[0397] Further, a method for preparing 4-amino4-oxo derivatives of theproducts of FIGS. 13d, 13 e, 13 f or 13 g of this invention includessubjecting these products to hydrolysis with 6N (six-normal solution)HCl for about six hours at room temperature.

[0398] Another embodiment of this invention is a method for preparing2,4-dioxo derivatives of the products of FIGS. 13d, 13 e, 13 f or 13 gthat includes subjecting these products to hydrolysis with 6N HCl undermild reflux conditions for about two hours.

Example 17

[0399] The following example provides a method for preparing6-(thiophenylmethyl)-2,4-diaminopyrido[2,3-d]pyrimidine (FIG. 14e) and6-(thionapthylmethyl)-2,4-diaminopyrido[2,3-d]pyrimidine (FIG. 14f)generally represented by formula 11. Reference letters and numeralscorrespond with those in FIG. 14.

2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxyaldehyde (FIG. 14 b)

[0400] About 2.0 g of the nitrile (FIG. 14a) was dissolved in about 60ml warm HCO₂H under N₂. About 10 g of damp Raney Ni was added. Themixture was refluxed for 2 hours and filtered through Celite®. Thefiltrate was concentrated under reduced pressure at a temperature of 50°C. with the aid of EtOH. The resulting viscous orange residue was thendissolved in about 150 ml of boiling H₂O. The boiling solution wastreated with Norit® and filtered through Celite® while hot. The filtratewas neutralized to pH 7 with 1 N NaOH to give a yellow precipitate. Thesuspension was refrigerated overnight, filtered and washed with H₂O,EtOH and Et₂O to yield about 1.75 g of a yellow solid. Examination byTLC (4:1:0.1 CHCl₃:MeOH:NH₄OH) showed a dominant UV-absorbing spot atR_(f)=0.38 and contamination spots at R_(f)=0.19 and at baseline. Thespot at R_(f)=0.19 was, after chromatographic separation, determined tocorrespond to the R_(f) value of compound (FIG. 14c).

2,4-diaminopyrido[2,3-d]pyrimidine-6-methanol (FIG. 14 c)

[0401] About 5.0 g of crude aldehyde (FIG. 14b) was pulverized, driedand stirred in anhydrous MeOH under N₂ overnight. About 0.17 g of NaBH₄was added to the mixture four times at intervals of 15 minutes. Themixture was stirred for 5 additional hours. Insoluble material wasfiltered and the filtrate was treated with about 200 ml H₂O. Thefiltrate was then concentrated under reduced pressure at a temperatureof 35° C. until a yellow precipitate began to form. The mixture was thenrefrigerated overnight, filtered and rinsed with H₂O, EtOH and Et₂O toyield about 1.50 g of a yellow solid. TLC (4:1:0.1 CHCL₃:MeOH:NH₄OH)showed a product spot at R_(f)=0.41 and a slight spot corresponding tothe starting material. Separation was carried out by chromatography withsilica gel.

6-(Bromomethyl)-2,4-diaminopyrido[2,3-d]pyrimidine (FIG. 14 d)

[0402] About 0.24 g of crude alcohol (FIG. 14c) was dried with P₂O₅ at110° C. under vacuum overnight and then added to about 10 ml ofanhydrous dioxane. The mixture was stirred in an ice bath while dry HBrgas was bubbled through for 15 minutes, after which the flask wasquickly stoppered. The mixture continued to stir and the alcoholdissolved after approximately ½ hour. The solution stirred for 24 hoursand was then added dropwise to stirred Et₂O under N₂ to give a yellowsuspension. The suspension was refrigerated overnight, filtered andimmediately dried with P₂O₅ under vacuum at 50° C. to yield about 45 mgof the compound represented by FIG. 14d.

6-(Thiophenylmethyl-2,4-diaminopyrido[2,3-d]pyrimidine (FIG. 14 e)

[0403] About 0.12 ml of phenylthiol was dissolved in about 10 ml DMACand added dropwise to about 0.25 g of the compound of FIG. 14d. About 1g of K₂CO₃ was added to the mixture to adjust the pH to approximately 8.After 1 hour, the compound of FIG. 14d was not detectable by TLC(3:1:0.1 CHCl₃:MeOH:NH₄OH). A product spot appeared at R_(f)=0.33 withcontamination spots at R_(f)=0.51 and at baseline. The solid wasfiltered and rinsed with H₂O, EtOH and Et₂O to yield about 22 mg of thecompound of FIG. 14e.

6-(Thionaphthylmethyl)-2,4-diaminopyrido[2,3-d]pyrimidine (FIG. 14 f)

[0404] About 0.07 g of napthylenethiol was dissolved in DMAC (15 mL) andadded dropwise to about 0.10 g of the compound of FIG. 14d. About 0.3 gof Na₂CO₃ was added and the color of the reaction mixture changed fromyellow to green. The reaction was monitored by TLC (4:1:0.1CHCl₃:MeOH:NH₄OH). A product spot occurred at R_(f)=0.5. After 3 hours,starting material was still present. Also, the yellow color returned.The reaction was run overnight. The pH was then checked and found to beslightly acidic. The solution was added dropwise to about 100 ml of 1 NNa₂CO₃. The suspension was stirred for 15 minutes and refrigerated for 4hours. The solid was filtered and rinsed with H₂O, EtOH and Et₂O toyield about 25 mg of the compound of FIG. 14f.

Example 18

[0405] The following are various methods for making various2,4-diamino-6-substituted-benzylamino pyrido[2,3-d]pyrimidines generallyrepresented by formula 11. Reference letters and numerals correspondwith those in FIG. 15. The synthesis of the desired compounds isachieved via the reductive amination of 2,4-diamino-6 aminopyrido[2,3-d]pyrimidine 2, with the appropriately substituted aldehydeas generally illustrated in FIG. 15.

2,4-diamino-6-nitropyrido[2,3-d]pyrimidine (FIG. 15 a)

[0406] About 1 equivalent of 2,4,6-triamino pyrimidine was suspended inabout 50 ml of refluxing absolute ethanol with stirring under anatmosphere of nitrogen. Concentrated HCl was added dropwise to effectsolution and as soon as solution occurred, about 1.2 equivalents ofnitromalonaldehyde was added all at once. Within 10 minutes, a thickreddish voluminous precipitate started forming. TLC analysis indicatedthe presence of a yellow spot corresponding to that of the desiredproduct along with staring materials. The reaction mixture was stirredat reflux for 3.5 hours, immediately diluted with 30 ml of water, cooledand neutralized with concentrated NH₄OH. The precipitate was collectedon a funnel and was washed repeatedly with water to remove unreactedtriamino pyrimidine to yield pure2,4-diamino-6-nitropyrido[2,3-d]pyrimidine.

Example 19

[0407] The following is a method of making 2,4-diamino-6-(anilinomethyl)pyrido[2,3-d]pyrimidines generally represented by formula 11. Referenceletters and numerals correspond with those in FIG. 16.

[0408] About 1 equivalent of2,4-diamino-pyrido[2,3-d]pyrimidine-6-carbonitrile (FIG. 16a) (achievedvia literature procedures) was dissolved in 80% acetic acid. To thissolution was added about 5 equivalents of Raney Nickel followedimmediately by about 1.5 equivalents of the appropriately substitutedaniline (FIG. 16b). The mixture was hydrogenated under atmosphericpressure and at room temperature for 6 hours. TLC analysis at the end ofthis period indicated the presence of a spot corresponding to thedesired product. The reaction mixture was filtered through Celite® andthe filtrate was evaporated to dryness to yield a reddish residue. Thisresidue was dissolved in warm absolute ethanol and then neutralized inthe cold with 1 N Na₂CO₃ dropwise with stirring to deposit the crudeproduct. This solid was collected by filtration and was washedrepeatedly with acetone and dissolved in a large volume of methanol;silica gel was added and the methanol stripped off to yield a dry plug.Column chromatography using CHCl₃:MeOH (5:1) as the eluant yielded puretarget compounds represented by FIG. 16c.

Example 20

[0409] The following example provides methods for makingpyrido[3,2-d]pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 17.

2,4-dioxo-6-methylpyrido[3,2-d]pyrimidine (FIG. 17 a)

[0410] About 20 g of 5-aminouracil, 80 ml of 20% HCl and 4 ml ofcrotonaldehyde were heated together under reflux for 1 hour. Thesolution was evaporated to dryness under rotary evaporation. Water wasadded to the residue so as to make the mixture just stirrable and thenit was triturated with ammonium hydroxide with strong stirring until thepH increased to 10-11. Stirring was continued for another 10 minutes.The precipitate was filtered and was washed with minimal methanol andthen chloroform and dried to yield about 17.58 g of the compound of FIG.17a.

6-(Acetoxymethyl)-2,4-dioxopyrido[3,2-d]pyrimidine (FIG. 17 b)

[0411] About 1.77 g of the compound of FIG. 17a in 50 ml of glacialacetic acid containing about 6.5 g of MCPBA (57-85%) was refluxed for 3hours. About 40 ml of acetic anhydride was added to the hot reactionmixture and the refluxing was continued for another 30 minutes. Theclear brown solution was evaporated to dryness and the solid was stirredwith about 100 ml ether and filtered. The solid was crystallized fromethanol to yield about 1.55 g of the compound of FIG. 17b.

6-(Acetoxymethyl)-2,4-dichloropyrido[3,2-d]primidine (FIG. 17 c)

[0412] About 1.5 g of the compound of FIG. 17c was refluxed with about38 ml of phosphoryl chloride containing about 2.5 ml of triethylaminefor 8 hours. The volume was reduced to about 5 ml by rotary evaporation.The dark syrup which resulted was poured into crushed ice. The coldsuspension was extracted with methylene chloride (3×50 ml) and washedwith cold water until the washing were neutral. The organic layer wasdried over anhydrous sodium sulfate and evaporated to dryness underrotary evaporation. The dark solid residue was stirred and refluxed withpetroleum ether (30-60° C.) and a suitable amount of decoloringcharcoal, and filtered through Celite®, which was repeated twice. Thecombined liquid solution was concentrated until the light yellow solidprecipitated out and was allowed to cool to room temperature and storedin a refrigerator for 2 hours. The crystallized solid was filtered anddried to yield about 0.86 g of the compound of FIG. 17c.

2,4-diamino-6-(hydroxymethyl)pyrido[3,2-d]pyrimidine (FIG. 17 d)

[0413] About 2.5 g of the compound of FIG. 17c was heated with 30 ml ofliquid ammonia in a sealed bomb at between about 150-170° C. for 18hours. After cooling to room temperature, the bomb was opened and theliquid ammonia was allowed to evaporate at room temperature. The solidwas crystallized from glacial acetic acid and a small amount of water toyield about 1.24 g of the compound of FIG. 17d.

2,4-diamino-6-(bromomethyl)pyrido[3,2-d]pyrimidine (FIG. 17 e)

[0414] A suspension of about 0.72 g of the compound of FIG. 17d in 12 mLdry THF was stirred for 8 hours with 1 ml of phosphorus tribromide. Theprecipitated solid was filtered, washed with cold 50% THF-Ether, anddried to give the compound of FIG. 17e. Because of the instability, thiscompound was not purified further. The ¹HNMR showed that the majority ofthe solid was the desired compound.

2,4-diamino-6-(paramethoxyanilinylmethyl)pyrido[3,2-d]pyrimidine (FIG.17 f)

[0415] To a suspension of about 3.5 mmol of the compound of FIG. 17e inanhydrous dimethylacetamide was added 0.92 g anisidine and 1.03 ganhydrous potassium. After the suspension was stirred for 2 days, almostall of the compound of FIG. 17e disappeared. The solvent DMAC wasremoved under diminished pressure. The solid residue was washed withmethanol three times and filtered. To the combined liquid was addedsilica gel and the methanol was evaporated to dryness. Separation toafford pure product of FIG. 17f was carried out by columnchromatography.

2,4-diamino-6-(phenylthiomethyl)pyrido[3,2-d]pyrimidine (FIG. 17 g)

[0416] To a suspension of about 2.5 mmol of the compound of FIG. 17g inanhydrous dimethylacetamide was added 1 mL thiophenol and 690 mganhydrous potassium. The suspension was stirred for 3 days. The solventDMAC was removed under diminished pressure. The solid residue was washedwith methanol three times and filtered. The combined liquid was added tosilica gel and then the methanol was evaporated to dryness. A smallamount of product was separated through a dry column. Aftercrystallization, about 5 mg of pure product of FIG. 17g was obtained.

2,4diamino-6-(naphathalinylmethyl)pyrido[3,2-d]pyrimidine (FIG. 17 h)

[0417] The procedure for making the compound of FIG. 17f was repeatedwith a reaction time of 5 days to yield the compound of FIG. 17h.

Example 21

[0418] The following example provides methods for making the tricyclicpyrimidine compounds generally represented by formula 10. Referenceletters and numerals correspond with those of FIG. 10.

N-butoxycarbonyl-4-pireridine (FIG. 10 b)

[0419] About 2 g of 4-piperidine hydrochloride monohydrate (FIG. 10a)was dissolved in about 30 ml of N,N-dimethylformamide at temperatures ofbetween about 110-115° C. The solution was cooled to room temperature,and to this solution was added about 2.6 g of triethyl amine and asolution containing about 3.06 g of ditertiarybutyl decarbonate in 10 mlDMF. The reaction was continued for about 24 hours at room temperature.The DMF was removed under reduced pressure. About 100 ml of water wasadded to the residue and the mixture was extracted with ethyl ether(2×100 ml), and the organic layer dried over anhydrous MgSO₄. The etherwas evaporated to dryness under reduced pressure to yield about 2.33 gof the compound of FIG. 10b.

3-bromo-4-piperidine hydrobromide (FIG. 10 c)

[0420] About 2.6 g of N-butoxycarbonyl-4-piperdone (FIG. 10b) wasdissolved in about 70 ml of chloroform; to this solution was added about2.08 g Br₂ over a period of about 30 minutes. The reaction was continuedfor about 2 hours at room temperature during which a white precipitateof the compound of FIG. 10c was formed. The reaction mixture wasfiltered and washed with chloroform ether to yield about 2.76 g of thecompound of FIG. 10c.

2,4-diamino-5,6,7,8-tetrahydro-7-pyrido[4′,3′:4,5′]furo[2,3-d]pyrimidine hydrobromide (FIG. 10 f)

[0421] A solution containing about 1.83 g of the compound of FIG. 10c inabout 10 ml of anhydrous DMF was added dropwise to a suspensioncontaining about 0.504 g of 2,4-diamino-6-hydroxypyrimidine in about 3ml of anhydrous DMF. The reaction became a clear solution after about 1hour; the solution was then left at room temperature for about 48 hours.The white precipitate which formed was collected by filtration and airdried to yield about 0.66 g of the compound of FIG. 10f.

2,4-diamino-5,6,7,8-tetrahydro-(7-butoxycarbonyl)pyrido[4′,3′:4,5]furo[2,3-d]pyrimidine hydrobromide (FIG. 10 e)

[0422] The filtrate obtained in preparing the compound of FIG. 10c wasdiluted with about 150 ml of chloroform and washed with water, saturatedsodium bicarbonate and brine. The organic layer was dried over anhydrousMgSO₄, and the chloroform was removed under reduced pressure to yield aviscous brown oil. The residue was dissolved immediately in about 5 mlof anhydrous DMF, and added to a suspension containing about 0.252 g of2,4-diamino-6-hydroxypyrimidine in anhydrous DMF. The reaction wascontinued for about 48 hours at room temperature. The DMF was removedunder reduced pressure, and the residue was dissolved in 50 ml ofmethanol; about 1.7 g of silica gel was added and the mixture wasevaporated to dryness under reduced pressure. About 50 ml of ether wasadded to the silica gel plug; the homogenous plug collected afterfiltration was air-dried and then placed on top of a dry silica gelcolumn (1.5 cm×10 cm) and gradiantly eluted with MeOH in CHCl₃.Fractions containing the compound of FIG. 10e were pooled and evaporatedto dryness under reduced pressure to yield 0.025 g of the compound.

2,4-diamino-5,6,7,8-tetrahydro-7-(benzyl)pyrido[4′,3′:4,5]furo[2,3-d]pyrimidine(FIG. 10 g)

[0423] About 0.46 g of the compound of FIG. 10f was suspended in 5 ml ofanhydrous DMSO. About 0.483 g of anhydrous potassium carbonate and 0.24g of benzyl bromide were added to the suspension; the reaction wascontinued for 24 hours at room temperature. The mixture was then dilutedwith about 30 ml of water and stirred for 24 hours at room temperature.The resulting precipitate was collected by filtration, washed withwater, acetone, ether and air-dried. The crude solid was dissolved in amixture of DMF:MeOH (1:5); about 1.2 g of silica gel was added and themixture was evaporated to dryness under reduced pressure. The resultingsilica gel plug was placed on a top of a dry silica gel column (1.5cm×10 cm) and gradiantly eluted with MeOH in CHCl₃. The fractionscontaining the compound of FIG. 10g were pooled and evaporated todryness under reduced pressure; the resulting solid was triturated withether and filtered to yield about 0.156 g of product.

2,4-diamino-5,6,7,8-tetrahydro-7-[(3′,4′,5′-trimethoxy)benzyl]pyrido[4′,3′:4.5]furo[2,3-d]pyrimide(FIG. 10 h)

[0424] The compound of FIG. 10h was prepared and purified in the samemanner as the compound of FIG. 10g, only using 3′,4′,5′-trimethoxybenzylchloride instead of benzyl bromide to yield about 0.12 g of the compoundof FIG. 10h as a yellow solid.

2,4-diamino-5,6,7,8-tetrahydro-7-[(3′,5′-dimethoxy)benzyl]pyrido[4′,3′:4,5]furo[2,3-d]pyrimide(FIG. 10 i)

[0425] The compound of FIG. 10i was prepared and purified in the samemanner as the compound of FIG. 10g, only using 3′,5′-dimethoxybenzylchloride instead of benzyl bromide to yield about 0.126 g of thecompound of FIG. 10i.

2,4-diamino-5,6,7,8-tetrahydro-7-[(2′,4′-dichloro)benzyl]pyrido[4′,3′:4,5]furo[2,3-d]pyrimide(FIG. 10 j)

[0426] The compound of FIG. 10j was prepared and purified in the samemanner as the compound of FIG. 10g, only using 2′,4′-dichlorobenzylchloride instead of benzyl bromide to yield about 0.151 g of thecompound of FIG. 10j.

2,4-diamino-5,6,7,8-tetrahydro-7-[(3′,4′-dichloro)benzyl]pyrido[4′,3′:4,5]furo[2,3-d]pyrimide(FIG. 10 k)

[0427] The compound of FIG. 10k was prepared and purified in the samemanner as the compound of FIG. 10g, only using 3′,4′-dichlorobenzylchloride instead of benzyl bromide to yield about 0.131 g of thecompound of FIG. 10k.

24-diamino-5,6,7,8-tetrahydro-7-[(2′,6′-dichloro)benzyl]pyrido[4′,3′:4,5]furo[2,3-d]pyrimide(FIG. 10 l)

[0428] The compound of FIG. 10l was prepared and purified in the samemanner as the compound of FIG. 10g, only using 2′,6′-dichlorobenzylchloride instead of benzyl bromide to yield about 0.093 g of thecompound of FIG. 10l.

2,4-diamino-5,6,7,8-tetrahydro-7-[(2′,4′-dichloro)benzyl]pyrido[4′,3′:4,5]furo[2,3-d]pyrimide(FIG. 10 m)

[0429] 2,4-Diamino-5,6,7,8-tetrahydro-7-[(4″-benzoyl)diethyl-L-glutamicacid)]pyrido[4′,3′:4,5]furo[2,3-d]pyrimidine was prepared and purifiedin the same manner as the compound of FIG. 10g, only using4′-(chloromethyl)benzoyl glutamic acid diethyl ester instead of benzylbromide. About 1.5 ml of 1 N NaOH was added to a solution containingabout 0.183 g of this compound in 10 ml methoxyethanol, and the solutionstirred at room temperature for about 24 hours. The ethoxyethanol wasevaporated under reduced pressure, the residue was dissolved in about 10ml of water and stirring continued for an additional 24 hours. Thesolution was cooled in an ice bath and the pH was adjusted to about 3.5via dropwise addition of 1 N HCl. The precipitate formed was collectedby filtration, washed with water, acetone and ether and air-dried toobtain about 0.160 g of the compound of FIG. 10m.

Example 22

[0430] The compound of FIG. 10m was tested for DHFR inhibition, asdiscussed above in Example I, against Pneumocystis carinii (Pc),Toxoplasmosis gondii (Tg), Macrobacterium avium (Ma) and rat liver (RL).Ma is an opportunistic infection in HIV infected patients. Table 20 setsforth the IC₅₀ values. TABLE 20 Inhibitory Concentrations (IC₅₀, microM)and Selectivity Ratios Compound Pc Tg RL RL/Pc RL/Tg M. avium 10 m 10.921.5 85.8 7.9 4 0.97

[0431] The compound of FIG. 10m showed a weak inhibitory activityagainst DHFR, but displayed promising selectivity ratios of 7.9 and 4against Pc DHFR and Tg DHFR, respectively, against RL DHFR. In addition,compound 10m showed significant inhibitory activity against Ma DHFR andhad a selectivity ratio of 88 versus RL DHFR.

[0432] In addition, the ability of the compound of FIG. 10m to functionas a substrate of human CCRF-CEM folylpolyglutamate synthetase wasassessed, as discussed above in

Example 4. Results are presented in Table 21.

[0433] TABLE 21 Activity of the Compound of FIG. 10 m as Substrate forCCRF-CEM Human Leukemia Cell Folylpolyglutamate Synthetase substrateK_(m), microM V_(max,rel) V_(max)/K_(m(rel)) n Aminopterin 4.8 ± 0.7 10.21 ± 0.04 6 10 m 6.2 ± 1.4 0.29 ± 0.05 0.06 ± 0.01 2

Example 23

[0434] The following example provides methods for makingpyrido[3,2-d]pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 11.

[0435] The compound of FIG. 11a was synthesized from2,6-dichloropyridine in two steps—nitration at the 3 position withconcentrated sulfuric acid and 90% nitric acid, followed by substitutionof the 2-chloro moiety with a cyano group using CuCN at 180° C. Thecompound of FIG. 11b was synthesized via a direct aromatic substitutionof FIG. 11a with arylamines, 3′,4′,5′-trimethoxybenzylamine, orN-methylarylamines in 2 ethoxyethanol at temperatures between about120-140° C. The compound of FIG. 11b was then reduced by iron in acidicconditions under reflux in methanol to yield the intermediate2-cyano-3-amino-6-substituted pyridines (FIG. 11c). Monomethylglycol wasadded to increase the solubility. The compound of FIG. 11c was condensedwith formamidine hydrochloride in dimethyl sulfone at temperaturesbetween about 120 and 150° C. to provide the compound of FIG. 11d.

Example 24

[0436] The following example provides methods for makingpyrido[3,2-d]pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 12.

[0437] The compound of FIG. 12a was reduced with iron powder, followedby condensation with chlorohydrochloride in DMSO at temperatures ofbetween about 120 and 150° C. to yield2,4-diamino-6-chloro-pyrido[3,2-d]pyrimidine (FIG. 12c). The compound ofFIG. 12c was subjected to a substitution reaction with substitutedthiophenols, to yield three arylthiol compounds (FIGS. 12f, 12 g, and 12h) generally represented by FIG. 12d. The oxidation of these compoundswith hydrogen peroxide in acetic acid provided three correspondingsulfonyl compounds with the general structure represented by FIG. 12e.

Example 25

[0438] The following example provides methods for makingpyrido[3,2-d]pyrimidine compounds generally represented by formula 11.Reference letters and numerals correspond with those of FIG. 18.

[0439] The compound of FIG. 18a was prepared from 2,6-dichloropyridineby methods known in the art. The 3-amino group of the compound of FIG.18a was protected with an acetyl group by reacting the compound withacetyl anhydride and triethylamine in the presence of DMAP under refluxconditions in methylene chloride for 20 hours Part of the startingmaterial was converted to the N,N-diacetylated amino derivative, whichwas in turn converted in situ into the compound of FIG. 18b when stirredin a saturated sodium bicarbonate solution at room temperature for about1.5 hours. The compound of FIG. 18b was then oxidated withtrifluroperacetic acid in methylene chloride at room temperature for 24hours to yield the compound of FIG. 18c, 2-amidylpyridine—N-oxide. Thecompound of FIG. 18c was then heated at about 140° C. with a guanidinecarbonate base in 2-ethoxyethanol for about one hour to yield thecompound of FIG. 18d, 6-chloro-2-methyl-4-oxo-pyrido[3,2-d]pyrimidine.The compound of FIG. 18d was then reacted with the sodium salt ofp-thiocresol in anhydrous DMF at 125° C. to yield thepyrido[3,2-d]pyrimidine compound of FIG. 18e.

[0440] Any compatible sulfur, oxygen or nitrogen containing nucleophilecan be reacted with the compound of FIG. 18d to yield the correspondingpyrido[3,2-d]pyrimidine compound.

Example 26

[0441] Reference numerals correspond with those of FIG. 19.

[0442] A 2-amino4-oxo-5-cyanopyrrolo[2,3-d]pyrimidine(III) was preparedaccording to the method reported by Migawa, et al., “A Two StepSynthesis of the Nucleoside q Precursor2-amino-5-cyanopyrrolo[2-3-d]pyrimidine-4-one” Syn. Comm. 26:3317(1996)which involved the synthesis of chloroformylacetonitrile (110) fromchloroacetonitrile (109) and methyl formate as shown in FIG. 19.Cyclocon-densation of compound 10 with 2,6-diamino-4-oxopyrimidineafforded to the key intermediate in 65-70% overall yield. Reductiveamination of the 5-nitrile with the appropriate anilines orethyl-p-aminobenzoyl-L-glutamate in the presence of Raney nickel andhydrogen at 50 psi for 5-6 hours afforded the desired analogues whichwere purified by column chromatography and were obtained in 20-62%yield. This yielded the product generally represented by compound 112.For the classical analogue, compound 407, which is the compound 112where R₂ is p-benzoyl-L-glutamate, saponification of thechromatographically purified ester afforded the desired product. Thismethod was used to synthesize compounds 407-415, which are generallyrepresented by compound 112 in FIG. 19 with the following substitutions:Compound R₂ 407 p-benzoyl-L-glutamate 408 2,5-diOMe phenyl 409 3,5-diOMephenyl 410 2,4-diOMe phenyl 411 3,4,5-triOMe phenyl 412 2,5-diCl phenyl413 3,5-diCl phenyl 414 2,4-diCl phenyl 415 3-Cl phenyl

Example 27

[0443] Compounds 407-415, prepared as described in Example 26, weretested for inhibition of tgDHFR, pcDHFR, rlDHFR and E-coli thymidylatesynthase (ecTS) according to the methods disclosed in Example 1. Theperformance of these compounds was measured against TMP. Results arepresented in Table 22. TABLE 22 Inhibitory Concentration (IC₅₀) ofNonclassical 5 In μM. Cpd. R₂ pcDHFR rlDHFR tgDHFR rl/tg ecTS 4082-5,diOMe phenyl >47 >47 2.2 >21 >160(21%) 409 3,5-diOMe phenyl >20 >202.6 >8 >160(0%) 410 2-4-diOMe phenyl >16 >16 7.1 >1.4 >160(8%) 4113,4,5-triOMe phenyl >22 >22 6.9 >3 >290(8%) 412 2,5-diCl phenyl >25 >250.66 >38 ND 413 3,5-diCl phenyl 66.2 >63 3 >21 ND 414 2,4-diClphenyl >15 >15 2.2 >7 ND 415 3-Cl phenyl >26 >26 3.5 >7 >180(26%) TMP —12.0 133.0 2.7 49.0 —

[0444] Results of testing of the classical analogue, compound 407, areshown in Table 23. As can be seen from Table 22, the nonclassicalanalogues, compounds 408-415, were micromolar inhibitors of tgDHFR withthe 2,5-dichlorophenyl analogue being the most potent (IC₅₀=0.66 μM).All the analogues were poorly inhibitory against pcDHFR, rlDHFR andecTS.

[0445] Compound 407 was a poor inhibitor of TS from L. casei, E. coli,P. carinii (46, >12, 5.8 μM respectively) and DHFR from L. casei and E.coli (6.9 and 23 μM respectively). The growth inhibitory potency ofcompound 407 was compared to that of methotrexate (MTX) in continuousexposure against CCRF-CEM human lymphoblastic leukemia and a series ofMTX resistant sublines. Compound 407 was only 5-fold less potent thanMTX against the parenteral cells. DHFR-overexpressing cells were 43-foldresistant to MTX, but only 2.5-fold resistant to compound 407,suggesting that DHFR is not the primary target of this agent. MTXtransport-defective cells are 160-fold resistant to MTX, but <4-foldresistant to compound 407. This suggests either that the mutationdecreasing the V_(max) of MTX transport in this cell line does notaffect compound 407 in a similar manner or that compound 407 primarilyuses a separate transport pathway. Polyglutamation of tight-bindingenzyme inhibitors, like MTX, is not required in continuous exposure;this accounts for the lack of resistance of polyglutamylation-defectiveR₃₀dm to MTX. However, for inhibitors where polyglutamylation isrequired for potent target inhibition (for example ZD1694), resistancemay become apparent for polyglutamylation-defective cell lines, even incontinuous exposure. The weak cross-resistance of R₃₀dm to compound 407suggest either that the parent drug is a potent target inhibitor or thatpolyglutamylation of this drug is highly efficient. TABLE 23 GrowthInhibition of CCRF-CEM Human Leukemia Cells And Its MTX-resistantSublines By MTX and Compound 407 Resistance Cell Line Mechanism EC50, nMCEM sensitive MTX Compound 407  15 ± 0 (n = 3)  79 ± (n = 3) R1 ↑ DHFR6445 ± 55 (n = 2) 200 ± 20 (n = 2) R2 ↓ transport 2350 ± 550 (n = 2) 310± 30 (n = 2) R30dm ↑ ↑ FPGS  16 ± 1 (n = 2) 120 ± 10 (n = 2)

Example 28

[0446] Reference numerals correspond with those in FIG. 20.

[0447] The syntheses of compounds 420-434 was carried out by anucleophilic displacement of the key intermediate2,4-diamino-5-chloromethylfuro[2,3-d]pyrimidine. (117) with theappropriate thiol or aniline as shown in FIG. 20. The intermediate (117)was obtained from 2,6-diamino-4-hydrooxypyrimidine and dichloroacetone.Purified 117 afforded the target compounds in 16-39% yield.Chromatographic purification was necessary for the final compounds,which was carried out on silica gel using 3% MeOH in CHCl₃.

[0448] As can be seen from FIG. 20, the product of this method has thegeneral formula of compound 4, as described throughout thespecification. Referring to formula 4, in these particular compounds, Xand Y are both NH₂, L and M are both carbon and the bond between L and Mis a double bond, R₄ is hydrogen, A is CH, R3 is H, and B, R1 and R2 areas illustrated below in Table 24. TABLE 24 Compound B R₁ R₂ 420 S —phenyl 421 S — 1-naphthyl 422 S — 2-naphthyl 423 N H 1-naphthyl 424 N H2-naphthyl 425 O — 2-naphthyl 426 N H 2-phenoxyphenyl 427 N H4-phenoxyphenyl 428 N H 2-phenylphenyl 429 N CH₃ 2-naphthyl 430 N H2,5-dichlorophenyl 431 N CH₃ 3,4-dichlorophenyl 432 N CH₃3,4,5-trichlorophenyl 433 N H 3-methoxyphenyl 434 N CH₃2,5-dimethoxyphenyl

[0449] Compounds 420-434 were prepared from compound 118 as describedbelow:

2-4-Diamino-5-[(phenylthio)methyl]furo[2,3-d]pyrimidine (420)

[0450] To a 100 ml three neck round bottom flask was added sequentiallyanhydrous DMSO (15 mL), thiophenol (0.85 g), crude 117 (0.75 g) andanhydrous potassium carbonate (1.03 g). The reaction mixture was stirredat room temperature for 2 h and then quenched with water (150 mL). Theresultant suspension was stirred at room temperature for 10 h andfiltered. The solid was extracted twice with MeOH (150 mL+100 mL) andthe extracts were mixed with silica gel (2 g). The mixture wasevaporated under reduced pressure and dried in vacuo. The residue waspowered and poured on top of a dry column of silica gel (40 g). Thecolumn was washed with MeOH in CHCl₃. Fractions corresponding to asingle spot (TLC) of the product were pooled and evaporated. The residuewas washed with ether and air dried to afford 0.17 g (16% for two steps)of compound 420 as a light pinkish solid.

2,4-Diamino-5-[(1-naphthylthio)methyl]furo[2,3-d]pyrimidine (421)

[0451] To a 50 ml three neck round bottom flask was added sequentiallyanhydrous DMSO (8 ml), 1-naphthylthiol (0.89 g), compound 117 (0.63 g)and anhydrous potassium carbonate (0.92 g). The reaction mixture wasstirred at room temperature for 2 h and then quenched with distilledwater (16 ml). The resultant suspension was stirred at room temperaturefor 4 h, cooled to 5° C. and filtered. The solid was washed with coldwater (2×5 ml) and then stirred in ether (20 ml) for 8 h. The suspensionobtained was filtered and the residue dissolved in acetic acid (40 ml).The solution was mixed with charcoal (0.1 g) and stirred at 40° C. for15 min. The mixture was filtered and the filtrate evaporated to dryness.The residue was washed thoroughly with MeOH to yield 0.44 g (29%) ofcompound 422 as a light yellow solid which was homogeneous on TLC.

2,4-Diamino-5-[(2-naphthylthio)methyl]furo[2,3-d]pyrimidine (422)

[0452] To a 50 ml three neck round bottom flask was added, in order,anhydrous DMSO (5 mL), 2-naphthalenethiol (1.12 g), crude 117 (1.39 g)and anhydrous potassium carbonate (0.97 g). The reaction mixture wasstirred at room temperature for 2 h and then quenched with distilledwater (50 ml). The resultant suspension was stirred at room temperaturefor 8 h, cooled to 5° C. and filtered. The solid was washed with coldwater (2×5 ml) and then extracted several times with MeOH (100 ml).Extracts with a major spot of the product on TLC were pooled and mixedwith silica gel (2 g). The mixture was evaporated under reduced pressureand then dried in vacuo. The residue was ground into a fine powder andpoured on top of a dry column of silica gel (40 g). The column waswashed with MeOH in CHCl₃:200 ml (0.5%), 100 ml (1%), 100 ml (2%). Thecolumn was then eluted with 3% MeOH in CHCl₃. Fractions corresponding toa single spot were also pooled and evaporated. The residue was washedthoroughly with ether to afford an additional 0.05 g of the pureproduct. Fractions with a major spot of the product and a faint spotjust below the product spot were also pooled and evaporated. The residuewas washed thoroughly with MeOH to afford an additional 0.06 g of thepure product (combined yield 10% for two steps) compound 422 as a lightpinkish solid.

2,4-Diamino-5-[(1-naphthylamino)methyl]furo[2,3-d]pyrimidine (423)

[0453] To a 50 ml three neck round bottom flask was added sequentiallyanhydrous DMSO (5 ml), 1-aminonaphthalene (0.66 g), Compound 117 (0.50g) and anhydrous potassium carbonate (0.69 g). The reaction mixture wasstirred at 50° C. for 8 h under nitrogen in the dark. The reaction wasthen quenched with distilled water (15 ml) and the resultant suspensionstirred at room temperature for 4 h, cooled to 5° C. and filtered. Theresidue was washed with cold water (2×5 ml) and then dissolved in hotMeOH, and the solution mixed with silica gel (2 g). The mixture wasevaporated under reduced pressure and then dried in vacuo. The residuewas ground into a fine powder and poured on top of a dry column ofsilica gel (40 g). The column was washed with MeOH in CHCl₃:200 ml(0.5%), 100 ml (1%), 100 ml (2%). The column then eluted with 3% MeOH inCHCl₃. Fractions corresponding to a single spot of the product (TLC)were pooled and evaporated. The residue was washed with ether and airdried to afford 0.25 g (33%) of compound 423 as a light yellow solid.

2-4-Diamino-5-[(2-naphthylamino)methyl]furo[2,3-d]pyrimidine (424)

[0454] To a 50 ml three neck round bottom flask was added, in sequence,anhydrous DMSO (4 mL) 2-aminonaphthalene (0.54 g), compound 117 (0.61 g)and anhydrous potassium carbonate (0.55 g). The reaction mixture wasstirred at 45° C. for 8 h under nitrogen in the dark and then quenchedwith distilled water (12 ml). The resultant suspension was stirred atroom temperature for 4 h, cooled to 5° C. and filtered. The solid waswashed with cold water (2×5 ml) and then dissolved in hot MeOH. Thesolution was then mixed with silica gel (2 g) and the mixture evaporatedunder reduced pressure and then dried in vacuo. The residue was groundinto a fine powder and poured on top of a dry column of silica gel (40g). The column was washed with MeOH in CHCl₃:200 ml (0.5%), 100 ml (1%),100 ml (2%). The column was then eluted with 3% MeOH in CHCl₃. Fractionscorresponding to a single spot of the product (TLC) were pooled andevaporated. The residue was washed with ether and air dried to afford0.27 g (29%) of compound 424 as a white solid.

2,4-Diamino-5-[(2-naphthoxy)methyl]furo[2,3-d]pyrimidine (425)

[0455] To a 50 ml three neck round bottom flask was added in sequenceanhydrous DMSO (4 ml), 2-naphthol (0.27 g), compound 117 (0.25 g) andanhydrous potassium carbonate (0.26 g). The reaction mixture was stirredat 45° C. for 24 h under nitrogen in the dark and then quenched withdistilled water (12 ml). The resultant suspension was stirred at roomtemperature for 4 h, cooled to 5° C. and filtered. The sold was washedwith cold water (2×5 ml) and then dissolved in MeOH. The solution wasmixed with silica gel (1 g) and the mixture was evaporated under reducedpressure and dried in vacuo. The residue was ground into a fine powderand poured on top of a wet column of silica gel (30 g) and eluted with3% MeOH in CHCl₃. Fractions corresponding to a single spot of theproduct (TLC) were pooled and evaporated to afford 0.09 g compound 425(23%) as a white solid.

2,4-Diamino-5-[(2′-phenoxyanilino)methyl]furo[2,3-d]pyrimidine (426)

[0456] To a 50 ml three neck round bottom flask was added in orderanhydrous DMSO (4 ml), 2-phenoxyaniline (0.35 g), compound 117 (0.25 g)and anhydrous potassium carbonate (0.21 g). The reaction mixture wasstirred at 45° C. for 8 h under nitrogen in the dark and then quenchedwith distilled water (12 ml). The resultant suspension was stirred atroom temperature for 4 h, cooled to 5° C. and filtered. The solid waswashed with cold water (2×5 ml) and then dissolved in MeOH. The solutionwas mixed with silica gel (1 g). The mixture was evaporated underreduced pressure and dried in vacuo. The residue was ground into a finepowder and poured on top of a wet column of silica gel (30 g) and elutedwith 3% MeOH in CHCl₃. Fractions corresponding to a single spot (TLC) ofthe product were pooled and evaporated. The residue was washed withether and dried to afford 1.16 g (37%) of compound 426 as a white solid.

2,4-Diamino-5-[(4′-phenoxyanilino)methyl[furo[2,3-d]pyrimidine (427)

[0457] Using a similar method to that for compound 426, 0.06 g ofchromatographically pure compound 427 was obtained as a white solid.Fractions corresponding to a major spot of the product (TLC) and a spotof some unknown impurity just below were pooled and evaporated. Theresidue was washed with MeOH to afford an additional 0. 12 g of theproduct which was homogeneous on TLC.

2,4-Diamino-5-[(2′-phenylanilino)methyl]furo[2,3-d]pyrimidine (428)

[0458] To a 50 ml three neck round bottom flask with compound 117 (0.30g), 2-aminobiphenyl (0.38 g) and K₂CO₃ (0.25 g) was added anhydrous DMSO(3 ml) and the reaction mixture stirred for 8 h at 45° C. under nitrogenin the dark. Distilled water (10 ml) was added to precipitate theproduct and the suspension was stirred for 2 h, cooled to 5° C. andfiltered. The solid was washed with cold water (2×2 ml) and suspended inMeOH (200 ml). Silica gel (1 g) was added and the suspension sonicatedand heated to 50° C. for 15 min, evaporated under reduced pressure anddried in vacuo for 10 h. The residue was powdered and poured on top of awet column of silica gel (30 g) and eluted with 3% MeOH in CHCl₃.Fractions corresponding to the product spot (TLC) were pooled andevaporated. The residue was washed with ether and air dried to afford0.24 g (48%) of compound 428 as a light yellow solid. Recrystallizationfrom MeOH afforded the analytically pure compound 428.

2,4-Diamino-5-[(N-methyl-2-naphthlamino)methyl]furo[2,3-d]pyrimidine(429)

[0459] To a suspension of compound 424 (0.10 g, 0.32 mmol), 30% (w/w)aqueous solution of formaldehyde (0.1 ml) and NaCN BH₃ (0.063 g) inacetonitrile (10 ml) was added dropwise concentrated hydrochloric aciduntil the suspension dissolved. The solution was stirred at roomtemperature for 1 h. The solvent was evaporated under reduced pressureand the residue dissolved in a minimum amount of distilled water and thepH of the solution adjusted to 7 with concentrated NH₄OH. The resultingsuspension was sonicated, cooled to 5° C. and filtered. The residue waswashed with cold water (2×3 ml), stirred in MeOH (5 ml) for 12 h andfiltered. The residue was washed with MeOH (2×3 ml) to afford 0.065 g(64%) of compound 429 as a light yellow solid.

2,4-Diamino-5-[(2′,5′-dichloroanilino)methyl]furo[2,3-d]pyrimidine (430)

[0460] To a 100 ml three neck round bottom flask was added, in sequence,anhydrous DMSO (30 ml), 2,5-dichloroaniline (2.43 g), crude compound 117(1.49 g) and anhydrous potassium carbonate (2.07 g). The reactionmixture was stirred at 45° C. for 8 h and then quenched with distilledwater (150 ml). The resultant suspension was stirred at room temperaturefor 8 h and filtered. The solid was extracted with MeOH (100 mL) severaltimes and extracts which gave a major spot of the product on TLC werepooled and mixed with silica gel (2 g) and the mixture evaporated underreduced pressure and then dried in vacuo. The residue was ground into afine powder and poured on top of a dry column of silica gel (40 g). Thecolumn was washed with MeOH in CHCl₃: 200 mL (0.5%), 100 mL (1%), 100 ml(2%). The column was then eluted with 3% MeOH in CHCl₃. Fractionscorresponding to a single spot of the product (TLC) were pooled andevaporated. The residue obtained was washed with ether and air dried toafford 0.067 g (3% for two steps) of compound 430 as a light yellowsolid.

2,4-Diamino-5-[(N-methyl-3′,4′-dichloroanilino)methyl]furo[2,3-d]pyrimidine(431)

[0461] To a 100 ml three neck round bottom flask was added anhydrousDMSO (30 ml), N-methyl-3,4-dichloroaniline (2.64 g), crude compound 117(1.49 g) and anhydrous potassium carbonate (2.07 g). The reactionmixture was stirred at 45° C. for 24 h and then quenched with distilledwater (150 ml). The resultant suspension was stirred at room temperaturefor 4 h and filtered. The residue was extracted with MeOH (100 mL)several times and extracts which gave a major spot of the product on TLCwere pooled and mixed with silica gel (2 g). The mixture was evaporatedunder reduced pressure and then dried in vacuo. The residue was groundinto a fine powder and poured on top of a dry column of silica gel (40g). The column was washed with MeOH in CHCl₃:200 mL (0.5%), 100 mL (1%),100 ml (2%). The column was then eluted with 3% MeOH in CHCl₃. Fractionscorresponding to a single spot (TLC) of the product were pooled andevaporated. The residue was washed with ether and air dried to afford0.08 g (3% for two steps) of compound 431 as a white solid.

2,4-Diamino-5-[(N-methyl-3′,4′,5′-trichloroanilino)methyl]furo[2,3-d]-pyrimidine (432)

[0462] To a 100 ml three neck round bottom flask was added3,4,5-trichloroaniline (1.96 g), potassium carbonate (1.66 g),acetonitrile (25 mL) and iodomethane (1.42 g). The reaction mixture wasstirred at 40° C. for 3 days under nitrogen in the dark. The reactionmixture was then quenched with ethyl acetate (50 ml) and filtered. Theresidue was washed with ethyl acetate (3×20 ml). The filtrate and thewashings were combined and washed with water (3×30 ml). The organiclayer was then filtered through anhydrous MgSO₄ and evaporated underreduced pressure. The residue was dissolved in a minimum amount of ethylacetate/hexane 1:12 and the solution was placed on a wet column ofsilica gel (80 g) and eluted with ethyl acetate/hexane 300 ml (1:12),500 ml (1:8) to afford 0.88 g of N-methyl-3,4,5-trichloraniline (42%) asa white solid. The product was used directly for the condensationwithout characterization. To a 100 ml three neck round bottom flask wasadded anhydrous DMSO (5 ml), N-methyl-3,4,5-trichloroaniline (0.88 g)crude compound 117 (0.20 g) and anhydrous potassium carbonate (0.56 g).The reaction mixture was stirred at 45° C. for 24 h and then quenchedwith distilled water (15 ml). The resulting suspension was stirred atroom temperature for 4 h and filtered. The residue was extracted withMeOH (100 ml) several times and the extracts which showed a major spotof the product on TLC were pooled and mixed with silica gel (2 g). Themixture was evaporated under reduced pressure and dried in vacuo. Theresidue was ground into fine powder and poured on top of a dry column ofsilica gel (40 g). The column was eluted with 3% MeOH in CHCl₃.Fractions corresponding to a single spot of the product were pooled andevaporated. The residue was washed with ether and air dried to afford0.06 g (16% for two steps) of compound 432 as a light yellow solid.

2,4-Diamino-5-[(3′-methoxyanilino)methyl]furo[2,3-d]-pyrimidine (433)

[0463] To a 100 ml three neck round bottom flask was added anhydrousDMSO (15 ml), 3-methoxyanilino (0.93 g), crude compound 117 (0.75 g) andanhydrous potassium carbonate (1.04 g). The reaction mixture was stirredat 45° C. for 8 h and then quenched with distilled water (150 ml). Theresulting suspension was stirred at room temperature for 8 h andfiltered. The residue was dissolved in MeOH and the solution was mixedwith silica gel (2 g). The mixture was evaporated under reduced pressureand dried in vacuo. The residue was ground into a fine powder and pouredon top of a dry column of silica gel (40 g). The column was washed with200 mL of 0.5%, 100 mL of 1%, 100 mL of 2% of MeOH in CHCl₃. The columnwas then eluted with 3% MeOH in CHCl₃. Fractions containing a singleproduct spot were pooled and evaporated. The residue was washed withether and air dried to afford 0.12 g (11% for two steps) of compound 433as a white solid.

2,4-Diamino-5-[(N-methyl-2′,5′dimethoxyanilino)methyl]furo[2,3-_(d)]pyrimidine(434)

[0464] To a 100 ml three neck round bottom flask was added2,5-dimethoxyaniline (1.53 g), anhydrous potassium carbonate (1.66 g),acetonitrile (25 ml) and iodomethane (1.42 g). The reaction mixture wasstirred at 40° C. for 3 days under nitrogen in the dark. The reactionmixture was quenched with ethyl acetate (50 ml) and filtered. Theresidue was washed with ethyl acetate (3×20 ml) The filtrate and thewashings were combined and washed with water (3×30 ml). The organiclayer was filtered through MgSO₄ and evaporated under reduced pressure.The residue was dissolved in a minimum amount of ethyl acetate/hexane1:8. The solution was placed on a wet column of silica gel (80 g) andeluted with ethyl acetate/hexane: 300 ml (1:8), 500 ml (1:5) to afford0.51 g of N-methyl-2,5-dimethoxyaniline. To a 50 ml three neck roundbottom flask with compound 117 (0.32 g, 1.61 mmol),N-methyl-2,5-dimethoxyaniline (0.40 g) and anhydrous potassium carbonate(0.27 g) was added anhydrous DMSO (3 ml) and the reaction mixturestirred at 45° C. for 8 h under nitrogen in the dark. Distilled water(10 ml) was added to precipitate the product and the suspension stirredfor 2 h and then cooled to 5° C. and filtered. The solid was washed withcold water (2×2 ml) suspended in MeOH (200 ml). Silica gel (1 g) wasadded and the suspension sonicated and then heated to 50° C. for 15 min.The suspension was then evaporated under reduced pressure and dried invacuo at room temperature for 10 h. The residue was powdered and pouredon top of a wet column of silica gel (40 g) and eluted with 5% of MeOHin CHCl₃. Fractions corresponding the product spot (TLC) were pooled andevaporated. The residue was washed with ether and air dried to afford0.15 g (28%) of compound 434 as a white solid.

Example 29

[0465] Compounds 420-434, prepared as described in Example 28, weretested for inhibiting DHFR according to the methods of Example 1. Theinhibitory concentration (IC₅₀) values along with the selectivity ratios(IC₅₀) rlDHRF/IC₅₀ pcDHRF or tgDHFR) for compounds 420-434 are listed inTable 23 along with epiroprim and TMP. Compound 422, the S-2-naphthyl,and compound 428, the NH-2-biphenyl, were extremely selective againstpcDHFR with selectivity ratios of 18.9 and 17.8, respectively. Compound422 was also significantly potent against pcDHFR (IC₅₀ of 0.66 μM).Compared to epiroprim and TMP, compound 422 is significantly more potentand more selective against pcDHFR. Compound 428 displayed highselectivity for pcDHFR at 17.8 with an IC₅₀ of 7.7 μM comparable toepiroprim and TMP in potency but with better selectivity. The otheranalogues were neither potent nor significantly selective. The resultssuggest that all three features, namely the heterocyclic ring, thebridge and the side chain aryl moiety combine to provide pcDHFRselectivity in the series. TABLE 25 Inhibitor Concentrations (IC₅₀, μM)and Selectivity Ratios of 5-Substituted Furo[2,3-d]pyrimidines AgainstpcDHFR, tgDHFR and rlDHFR. (5,8) pcDHFR rlDHFR rl/pc tgDHFR rl/tg420 >26 252 ND >26 ND 421 19 23 1.2 19 1.2 422 0.65 12.3 18.9 11.6 1.1423 13.5 12 0.89 37 0.32 424 41 36.5 0.89 38 0.96 425 14 60.3 4.31 >42ND 426 >12 >12 ND >12 ND 427 8.1 16.2 2.00 32.4 0.50 428 7.7 187 17.7945.4 3.02 429 14.8 1406 0.99 23.6 0.62 430 50.9 71.9 1.4 >47 ND 43144.8 >27 ND >27 ND 432 284 34.3 0.1 21.5 1.6 433 >31.3 >31.3 ND >31.3 ND434 >27 >27 ND >27 ND TMP 12 133 11.1 2.7 49 Epiroprim 2.6 33.2 12.80.48 70.6

Example 30

[0466] Compounds having the general formula 14, as more specificallyidentified below, were evaluated as inhibitors of DHFR in the mannerdescribed above in Example 1, except that the compounds were notcompared with TMQ, PTX, TMP or MTX. Compound numbers correspond withthose described in Table 5 above.

[0467] Table 26 below illustrates the results of the testing. TABLE 26Inhibitory Concentrations (IC₅₀ μM) and Selectivity Ratios SelectivityRatio: Selectivity RL Ratio: Pc RL DHFR/Pc RL Compound # DHFR¹ DHFR¹DHFR Tg DHFR¹ DHFR/Tg 113-102 38 97 2.55 17% @ 75 μM ND 113-103 28% @6.4 ND 6.3 1.00 28 μM 113-118 60% @ 48% @ ND 15% @ 31 μM ND 31 μM 31 μM113-125  0% @ 49% @ ND 12.2 ND 11 μM 11 μM 113-143 51% @ 39% @ ND 34% @61 μM ND 61 μM 61 μM 113-143B 18% @ 3.7 <0.11 6 0.62 40 μM 113-148 45% @27% @ ND 27% @ 85 μM ND 85 μM 85 μM 113-154B 36 1.8 0.05 4.4 0.41113-149 10% @ 13% @ ND 13% @ 35 μM ND 35 μM 35 μM 113-154A 55 2.3 0.0410.2 0.23

[0468] As noted in the table, some of the inhibition did not reach 50%.For those results, the % inhibition and the concentration at which this% was achieved are noted. As can be seen from the above table, thesecompounds would have a relatively low toxicity to mammals.

Example 31

[0469] Compounds having the general formula 14, as specificallyidentified below, were evaluated as inhibitors of DHFR and TS asgenerally described in Examples 6-8. Some of the compounds were comparedwith TMP and MTX and/or ZD1694. All of the compounds were also comparedwith LY231514, which is a thymidylate synthase inhibitor. Results arepresented in Tables 27 and 28 below. TABLE 27 Inhibition ofDihydrofolate Reductase (IC₅₀ in μM) Compound Molecular Weight L. casei113-102 348  >2.9 × 10⁻⁵ (17%)* 113-103 254    3.9 × 10⁻⁶ 113-118 395 >2.5 × 10⁻⁵ (0%) 113-125 292    3.4 × 10⁻⁶ 113-143 402  >2.5 × 10⁻⁵(8%) 113-148 372    2.7 × 10⁻⁵ 113-154B 289    3.5 × 10⁻⁶ 113-149 445 >2.3 × 10⁻⁵ (8%) 113-154A 342    2.9 × 10⁻⁶ 113-161 426    2.4 × 10⁻⁸LY231514** 427    2.3 × 10⁻⁴ trimethoprim 290    3.5 × 10⁻⁷ methotrexate454   1.10 × 10⁻⁸

[0470] TABLE 28 Inhibition of thymidylate synthase (IC₅₀ in μM) CompoundMolecular Weight L. casei 113-161 426 9.8 × 10⁻⁶ LY231514** 427 2.1 ×10⁻⁵ ZD 1694*** 458 8.0 × 10⁻⁶

Example 32

[0471] Compound 113-161 was also tested for its ability to inhibit thegrowth of FaDu human squamous cell carcinoma cell lines. Performance wastested against MTX using hypoxanthine (Hx) and Thymidine (TdR). Resultsare shown in FIG. 21. The protection study indicates that 113-161 almostcompletely reversed the TS inhibition unlike MTX, and almost completelyreversed DHFR and TS when both were supplied.

[0472] Protection after 120 hours with di-LV was also determined.Results are shown in FIG. 22. All of the tested compounds reversedinhibition with leucovorin.

Example 33

[0473] The compounds identified in Table 29 below were further testedfor their in vitro anticancer activity by the National Cancer Institute.GI₅₀ represents the concentration at which growth was inhibited by 50%.TABLE 29 In Vitro Anti-Cancer Activity Panel/ Cell 113-102 113-103113-118 113-125 113-143 113-148 Line GI₅₀ GI₅₀ GI₅₀ GI₅₀ GI₅₀ GI₅₀113-154B GI₅₀ 113-149 GI₅₀ 113-154A GI₅₀ Leukemia CCRF-CEM 4.72E−061.33E−05 >1.00E−04   >1.00E−04   >1.00E−04   2.97E−06 2.42E−05 5.80E−08HL-60 (TB) 2.01E−05 >1.00E−04   >1.00E−04   3.95E−05 4.81E−06 2.38E−056.47E−08 4.18E−05 K-562 >1.00E−04   4.85E−05 3.38E−05 8.60E−06 5.91E−062.43E−05 8.46E−08 2.30E−05 MOLT-4 >1.00E−04   2.06E−05 1.73E−05 8.07E−067.46E−06 1.93E−05 2.85E−07 3.87E−05 RPMI-8226 >1.00E−04  6.02E−05 >1.00E−04   7.16E−05 7.67E−05 4.39E−07 6.74E−05 SR >1.00E−04  5.95E−05 3.16E−05 4.91E−06 3.51E−06 2.43E−05 3.14E−08 3.25E−05 Non-SmallCell Lung Cancer A549/ATCC >1.00E−04   2.96E−05 9.98E−055.43E−05 >1.00E−04   6.46E−06 2.12E−05 1.73E−07 2.61E−05 EKVX 6.45E−069.35E−08 7.09E−05 >1.00E−04   5.78E−06 9.27E−07 4.24E−07 5.21E−06 HOP-921.60E−05 6.34E−05 3.10E−05 2.41E−05 3.16E−05 1.63E−05 4.00E−05 4.67E−071.82E−05NCI-H226 >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04  1.24E−05 3.54E−05 1.95E−07 2.30E−05 NCI-H23 >1.00E−04   >1.00E−04  9.73E−05 >1.00E−04   6.66E−05 8.47E−06 3.68E−05 2.78E−07 2.69E−05NCI-H322M >1.00E−04   5.99E−05 7.47E−05 >1.00E−04   >1.00E−04  1.01E−05 >1.00E−04   5.81E−07 2.69E−05 NCI-H460 >1.00E−04   3.45E−054.25E−05 6.02E−05 >1.00E−04   7.70E−06 1.39E−05 1.62E−07 1.93E−05NCI-H522 >1.00E−04   8.40E−05 2.91E−05 3.44E−05 6.24E−06 8.34E−063.38E−05 4.77E−08 2.50E−05 Colon Cancer COLO 205 >1.00E−04   6.65E−053.82E−05 >1.00E−04   3.44E−05 1.17E−05 5.99E−05 1.90E−07 4.82E−05HCC-2998 >1.00E−04   6.90E−05 1.74E−05 3.17E−07 HCT-116 >1.00E−04  3.60E−05 4.83E−05 >1.00E−04   >1.00E−04   4.51E−06 3.19E−05 2.86E−073.68E−05 HCT-15 2.52E−05 5.53E−05 5.14E−05 5.79E−05 4.89E−05 1.02.E−052.04E−05 1.27E−07 2.43E−05 HT29 >1.00E−04   3.67E−054.84E−05 >1.00E−04   >1.00E−04   4.77E−06 3.84E−05 2.81E−07 1.00E−05KM12 >1.00E−04   5.06E−05 4.84E−05 >1.00E−04   >1.00E−04   1.22E−053.47E−05 1.94E−07 4.65E−05 SW-6204.16E−05 >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04   1.71E−057.34E−05 2.36E−07 CNS Cancer SF-268 >1.00E−04   >1.00E−04   >1.00E−04  3.71E−05 >1.00E−04   1.10E−05 4.55E−05 6.69E−07 3.95E−05SF-295 >1.00E−04   5.12E−05 7.93E−05 4.97E−05 1.78E−05 8.43E−06 3.51E−054.18E−08 3.66E−05 SF-539 >1.00E−04   >1.00E−04   3.02E−05 2.18E−053.39E−05 7.20E−06 3.80E−05 7.45E−08 2.04E−05SNB-19 >1.00E−04   >1.00E−04   >1.00E−04   3.74E−05 >1.00E−04   1.09E−057.04E−05 2.73E−07 6.74E−05 SNB-75 >1.00E−04   7.38E−08 >1.00E−04  2.42E−05 >1.00E−04   1.76E−05 3.62E−05 3.58E−07 2.75E−05 U251 1.22E−054.40E−05 3.00E−05 3.11E−06 1.04E−05 2.95E−05 1.44E−07 2.34E−05 MelanomaLOX IMVI >1.00E−04   5.13E−05 5.00E−05 7.31E−05 2.59E−05 1.37E−054.10E−05 2.50E−07 3.00E−05 MALME- >1.00E−04   >1.00E−04  1.95E−05 >1.00E−04   3.96E−05 1.86E−05 4.69E−05 2.83E−07 4.74E−05 3MM14 >1.00E−04   4.83E−05 9.36E−05 >1.00E−04   >1.00E−04   1.20E−055.40E−05 7.70E−08 2.98E−05SK-MEL-2 >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04  1.31E−05 >1.00E−04   3.81E−07 9.83E−05 SK-MEL-283.93E−05 >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04  1.57E−05 >1.00E−04   1.15E−05 >1.00E−05 SK-MEL-5 >1.00E−04  6.91E−05 >1.00E−04   1.28E−05 5.80E−06 2.95E−05 6.11E−08 3.73E−05UACC-257 3.14E−05 5.42E−05 >1.00E−04   9.09E−06 6.36E−06 3.81E−058.02E−08 5.64E−05 UACC-62 7.94E−05 >1.00E−04  8.00E−05 >1.00E−04   >1.00E−04   1.23E−05 3.20E−05 3.99E−08 1.96E−05Ovarian Cancer IGROV1 >1.00E−04   5.99E−056.82E−05 >1.00E−04   >1.00E−04   1.42E−05 3.63E−05 6.11E−08 1.73E−05OVCAR-3 >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04  1.48E−05 8.33E−05 2.88E−08 7.48E−05 OVCAR-4 5.63E−05 8.70E−058.01E−05 >1.00E−04   >1.00E−04   8.88E−06 4.79E−05 8.21E−07 7.78E−05OVCAR-5 >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04  1.08E−05 >1.00E−04   2.60E−06 OVCAR-8 >1.00E−04   6.56E−05 5.25E−056.44E−05 >1.00E−04   1.76E−05 4.02E−05 3.64E−07 3.11E−05SK-OV-3 >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04   >1.00E−04  1.73E−05 7.68E−05 5.06E−07 8.88E−05 Renal Cancer 786-0 >1.00E−04  3.98E−05 >1.00E−04   4.45E−05 >1.00E−04   1.16E−05 3.63E−05 2.54E−073.28E−05 A498 1.43E−05 >1.00E−04   1.42E−05 5.28E−05 7.02E−08 1.94E−061.34E−05 1.36E−07 8.45E−07 ACHN 4.40E−05 >1.00E−04  3.48E−05 >1.00E−04   6.26E−06 7.99E−06 3.75E−05 7.28E−07 3.57E−05 CAKI-18.79E−05 8.35E−05 >1.00E−04   3.02E−05 >1.00E−04   1.16E−05 5.57E−053.66E−07 4.95E−05 RXF 393 4.81E−06 1.83E−05 9.47E−052.56E−05 >1.00E−04   1.11E−05 3.02E−05 1.34E−07 3.27E−05SN12C >1.00E−04   2.73E−05 >1.00E−04   >1.00E−04   >1.00E−04   1.33E−055.80E−05 5.37E−07 4.31E−05 TK-10 >1.00E−04  8.87E−05 >1.00E−04   >1.00E−04   >1.00E−04   1.02E−05 5.51E−05 9.61E−075.89E−05 UO-31 >1.00E−04   2.19E−05 >1.00E−04   4.81E−05 >1.00E−04  4.78E−06 2.94E−05 3.47E−07 3.40E−05 Prostate Cancer PC-3 3.81E−053.33E−05 6.40E−05 >1.00E−04   >1.00E−04   9.47E−06 2.78E−055 4.79E−082.22E−05 DU-145 >1.00E−04   1.75E−05 >1.00E−04   >1.00E−04   >1.00E−04  1.31E−05 5.53E−05 2.24E−07 7.56E−05 Breast Cancer MCF75.00E−05 >1.00E−04   2.96E−05 6.19E−05 4.39E−05 1.26E−05 3.31E−051.18E−07 2.72E−05 NCI/ 2.23E−06 567E−05 7.44E−05 4.22E−06 6.75E−061.10E−05 1.45E−07 2.59E−05 ADRRES MDA-MB- 5.22E−05 >1.00E−04  <1.00E−04   9.10E−05 >1.00E−04   1.08E−05 8.31E−05 5.95E−07 3.73E−05231/ATCC HS 578T >1.00E−04   5.18E−06 <1.00E−04   8.07E−05 >1.00E−04  1.49E−05 2.23E−05 2.83E−07 2.70E−05 MDA-MB- >1.00E−04   >1.00E−04  9.54E−05 5.91E−05 >1.00E−04   1.30E−05 5.59E−05 1.01E−08 3.92E−05 435MDA-N >1.00E−04   4.11E−05 7.83E−05 7.55E−05 >1.00E−04   1.47E−054.65E−05 <1.00E−08   3.53E−05 BT-549 >1.00E−04   >1.00E−04   4.09E−055.21E−05 >1.00E−04   1.58E−05 1.86E−05 7.80E−06 1.93E−05 T-47D 5.04E−079.82E−06 6.64E−05 3.84E−06 3.10E−05 5.63E−07 6.02E−05

[0474] As can be seen from Table 29, several of the compounds were veryselective against various cell lines. For example, compound 113-102 wasvery selective against the CCRF-CEM leukemia cell line, the RXF-393renal cancer cell line and the NCI/ADR-RES breast cancer cell line.Compound 113-103 was selective against the EKVX non-small cell lungcancer and the SNB-75 CNS cancer cell lines. Compound 113-143 was verypotent against the A498 renal cancer cell line and also showed highselectivity against other cell lines (K-562, MOLTE, SR, NCI-H522, U251,UACC-257, ACHN, and NCI/ADR-RES). Compound 113-154B is selective againstEKUX non-small cell lung cancer, and compound 113-149 has selectivityagainst numerous cell lines. Compound 113-154A is selective against EKUXnon-small lung cancer and A498 renal cancer cell lines.

Example 34

[0475] Compounds having the general formula 15, as specificallyidentified below, were tested for their ability to inhibit DHFR asgenerally described in Example 1. Compound numbers correspond with thosedescribed in Table 3. Results are presented in Table 30 below. TABLE 30Inhibitory Concentrations (IC₅₀ μM) and Selectivity Ratios SelectivitySelectivity Ratio: Ratio: RL RL Compound DHFR/Pc DHFR/Tg No. Pc DHFR¹ RLDHFR¹ DHFR Tg DHFR¹ DHFR 111-157B 50% @ 51 μM 37% @ 51 μM ND 15.4 >3.3111-178 30% @ 14 μM  0% @ 14 μM ND 15% @ 14 μM ND 111-183 38% @ 29 μM10% @ 29 μM ND 38% @ 29 μM ND 111-184 36.2 40% @ 10 μM ND 27.7 ND111-190 10.3 20% @ 32 μM >3.2 38% @ 32 μM ND 111-191 16.2 12.6 0.78 4.52.80 111-192  8% @ 12 μM 34% @ 13 μM ND 63 ND 111-194 57% @ 63 μM 34% @63 μM ND 29% @ 63 μM ND 111-204 193 12% @ 38 μM >2 31% @ 38 μM ND

[0476] Compounds 111-190 and 111-204 showed high selectivity against ratliver DHFR.

Example 35

[0477] A compound corresponding to general formula 14 was synthesizedusing the following method. For compound 23m, synthesized according tothe following method, R₁₈ is CH₃, R₁₃ is H, Z₅ is R₁₄ which is H, A₁ isCH, B₁ is CH₁, R₁₅ is H, R₁₆ is H, and R₁₇ is p-benzoyl-L-glutamate.

[0478] Reagents and conditions used in the synthesis of the compound areas follows. Letters correspond with those shown in FIG. 23. a) guanidinecarbonate, EtOH, triethylamine, reflux, 69%. b) SOCl₂, reflux, 68%. c)benzylamine, triethylamine, n-BuOH, 90° C., 50%. d) MnO₂, 1,4-dioxane,reflux, 45%. e) Na/liq. NH₃, −78° C., 70%. f) PivCl, pyridine, reflux,75%. g) NIS, dark, rt, 52%. h) trimethylsilylacetylene,tetrakis(triphenylphosphine)palladium(O), CuI, triethylamine, THF, rt.i) n-Bu₄NF, THF, rt, 60%, two steps. j) diethyl4-iodobenzoyl-L-glutamate, tetrakis(triphenylphosphine)palladium(O),CuI, triethylamine, THF, rt, 42%. k) 5% Pd/C, H₂, 50 psi., 70%. 1) 1NNaOH, MeOH, 50° C., 71%.

[0479] The synthesis of target compound 23m (as described above) wasinitiated from 2-acetylbutyrolactone 23a which was used to synthesize5-(2-hydroxyethyl)-6-methyl-2-thiouracil according to the methodsgenerally taught by Badaway, J. Heterocyclic Chem. 33:229 (1996). Thismethod was modified to access a new synthetic pathway for theintermediate 23d. Compound 23a and guanidine carbonate were refluxedwith absolute ethanol in the presence of triethylamine to give 23b in69% yield. Compound 23b was converted to 23c in 68% yield by refluxingwith phosphorus oxychloride. The condensation step was carried out byrefluxing a mixture of 23c, benzylamine and triethylamine in n-BuOH,which afforded 23d in 50% yield. MnO₂ can be used to obtain 23e inreasonable yield (45%).

[0480] Dissolving Na/liq. NH₃ conditions were used to afford the product23f in good yield (70%) from compound 23e. These methods are generallydescribed by Hoops, et al., J. Heterocyclic Chem. 33:767 (1996) andMeade and Beuchamp, J. Heterocyclic Chem. 33:303 (1996).

[0481] The 2-amino group of intermediate 23f was protected with pivaloylchloride, which both increases the solubility of 23g and controls theregioselectivity of iodination in the subsequent step. Compound 23h wasthe only product obtained using NIS as the iodinating agent. Coupling of23h with trimethylsilylacetylene in the presence oftetrakis(triphenylphosphine)palladium(O), copper(I) iodide andtriethylamine in THF afforded compound 23i following the proceduregenerally taught by Shih and Gossett, Heterocycles, 36:825 (1993). Thesilyl protecting group of 23i was then removed by fluoide ion to givecompound 23j (60% yield overall in two steps). Compound 23j was thencoupled successfully with diethyl 4-iodobenzoyl-L-glutamate, undersimilar palladium-catalyzed reaction conditions as were employed inconverting 23h to 23i, to give compound 23k in 43% yield. Subsequentcatalytic hydrogenation (5% Pd—C/H₂/50 psi) and saponification affordedtarget compound 23m.

Example 36

[0482] The compounds identified below were further tested for their invitro anti-cancer activity by the National Cancer Institute. Results arepresented in Table 31 below. GI₅₀ represents the concentration at whichgrowth was inhibited by 50%. TABLE 31 In Vitro Anti-Cancer ActivityPanel/ Cell Line 111-157B 111-178 111-183 111-184 111-190 111-191111-192 111-194 111-204 Leukemia CCRF-CEM 1.66E−05 1.31E−051.00E−05 >1.00E−04   6.89E−05 4.06E−05 2.54E−05 >1.00E−04   6.54E−05HL-60 (TB) 1.59E−05 1.64E−05 1.82E−05 >1.00E−04   4.52E−05 >1.00E−04  K-562 3.38E−05 3.70E−05 2.58E−05 >1.00E−04   1.88E−05 >1.00E−04  3.03E−05 >1.00E−04   >1.00E−04   MOLT-4 2.06E−05 1.73E−051.03E−05 >1.00E−04   2.92E−05 3.12E−05 2.61E−05 4.52E−05 >1.00E−04  RPMI-8226 2.98E−05 1.07E−05 2.82E−05 >1.00E−04   4.69E−05 2.53E−052.76E−05 6.96E−05 >1.00E−04   SR 8.36E−06 1.56E−05 9.52E−06 >1.00E−04  5.02E−05 1.19E−05 7.14E−05 1.44E−05 5.48E−05 Non-Small Cell Lung CancerA549/ATCC 1.52E−05 1.41E−05 1.51E−05 >1.00E−04   2.73E−05 2.06E−052.73E−05 3.40E−05 >1.00E−04   EKVX 1.45E−05 2.93E−052.74E−05 >1.00E−04   1.67E−05 >1.00E−04  2.11E−05 >1.00E−04   >1.00E−04   HOP-62 1.24E−05 1.36E−054.36E−06 >1.00E−04   1.70E−05 3.72E−06 3.67E−05 3.20E−05 2.08E−05 HOP-921.46E−05 1.68E−05 1.08E−05 >1.00E−04   1.93E−05 9.38E−06 1.85E−056.57E−05 2.21E−05 NCI-H226 1.53E−05 2.99E−05 >1.00E−04   >1.00E−04  3.00E−05 >1.00E−04   2.96E−05 >1.00E−04   >1.00E−04   NCI-H23 1.75E−051.57E−05 2.70E−05 >1.00E−04   3.55E−05 >1.00E−04  2.93E−05 >1.00E−04   >1.00E−04   NCI-H322M 1.44E−05 3.36E−052.66E−05 >1.00E−04   1.99E−05 >1.00E−04   2.71E−05 7.12E−05 6.92E−05NCI-H460 1.37E−05 7.51E−06 5.41E−06 >1.00E−04   1.28E−05 5.12E−062.23E−05 3.92E−05 >1.00E−04   NCI-H522 1.47E−05 1.54E−056.14E−05 >1.00E−04   3.65E−05 >1.00E−04   2.31E−05 >1.00E−04   1.00E−04Colon Cancer COLO 205 3.02E−05 3.74E−05 >1.00E−04   >1.00E−04  7.17E−05 >1.00E−04   1.86E−05 >1.00E−04   8.37E−05 HCC-2998 1.91E−051.04E−05 2.54E−05 >1.00E−04   2.13E−05 >1.00E−04  3.76E−05 >1.00E−04   >1.00E−04   HCT-116 1.68E−05 2.03E−052.88E−05 >1.00E−04   3.00E−05 >1.00E−04   2.47E−05 4.19E−05 >1.00E−04  HCT-15 3.05E−05 9.31E−06 4.39E−06 >1.00E−04   8.39E−05 >1.00E−04  3.35E−05 >1.00E−04   >1.00E−04   HT29 2.46E−05 2.40E−052.22E−05 >1.00E−04   2.31E−05 >1.00E−04  2.59E−05 >1.00E−04   >1.00E−04   KM12 2.65E−05 3.10E−053.15E−05 >1.00E−04   4.78E−05 >1.00E−04  1.71E−05 >1.00E−04   >1.00E−04   SW-620 2.56E−05 1.73E−052.89E−05 >1.00E−04   5.18E−05 6.46E−05 4.91E−05 >1.00E−04   >1.00E−04  CNS Cancer SF-268 1.49E−05 2.95E−05 1.05E−05 >1.00E−04   1.24E−058.78E−06 2.19E−05 5.81E−05 6.29E−05 SF-295 1.90E−05 1.43E−051.85E−05 >1.00E−04   1.92E−05 4.62E−05 4.21E−05 4.08E−05 7.09E−05 SF-5392.23E−05 2.57E−05 4.86E−05 >1.00E−04   2.92E−05 7.01E−05 8.79E−053.07E−05 >1.00E−04   SNB-19 1.31E−05 2.53E−05 1.68E−05 >1.00E−04  2.63E−05 >1.00E−04   4.51E−05 5.48E−05 >1.00E−04   SNB-75 3.61E−067.87E−06 7.07E−06 >1.00E−04   7.90E−06 3.36E−06 2.57E−05 1.49E−052.01E−05 U251 1.45E−05 1.63E−05 1.22E−05 >1.00E−04   1.54E−05 9.15E−063.25E−05 3.07E−05 3.14E−05 Melanoma LOX IMVI 1.66E−05 2.43E−059.24E−06 >1.00E−04   2.70E−05 5.73E−05 1.77E−05 5.98E−05 >1.00E−04  MALME- 6.74E−06 1.80E−05 1.12E−05 >1.00E−04   6.74E−06 5.07E−061.01E−05 >1.00E−04   >1.00E−04   3M M14 2.59E−05 3.16E−054.51E−05 >1.00E−04   5.83E−05 >1.00E−04  1.96E−05 >1.00E−04   >1.00E−04   SK-MEL-2 2.99E−053.41E−05 >1.00E−04   >1.00E−04   2.30E−05 >1.00E−04  2.08E−05 >1.00E−04   2.34E−05 SK-MEL-28 2.81E−05 1.96E−053.40E−05 >1.00E−04   2.91E−05 >1.00E−04   3.37E−05 >1.00E−04   9.38E−05SK-MEL-5 2.05E−05 2.90E−05 7.52E−05 >1.00E−04   4.81E−05 >1.00E−04  1.55E−05 >1.00E−04   9.45E−05 UACC-257 1.83E−05 2.20E−056.98E−05 >1.00E−04   1.96E−05 >1.00E−04  1.97E−05 >1.00E−04   >1.00E−04   UACC-62 1.67E−05 1.23E−052.94E−05 >1.00E−04   1.89E−05 >1.00E−04   1.62E−05 >1.00E−04   8.51E−05Ovarian Cancer IGROV1 1.75E−05 1.86E−05 1.52E−05 >1.00E−04  1.73E−05 >1.00E−04   1.65E−05 >1.00E−04   3.96E−05 OVCAR-3 1.65E−051.77E−05 2.55E−05 >1.00E−04   2.36E−05 4.40E−053.57E−05 >1.00E−04   >1.00E−04   OVCAR-4 1.14E−05 9.64E−066.46E−06 >1.00E−04   8.96E−06 2.56E−05 >1.00E−04   >1.00E−04   OVCAR-53.11E−05 4.64E−05 >1.00E−04   >1.00E−04   3.12E−05 >1.00E−04  3.44E−05 >1.00E−04   >1.00E−04   OVCAR-8 1.48E−05 1.19E−056.05E−06 >1.00E−04   1.08E−05 4.10E−06 2.62E−05 3.52E−05 6.00E−05SK-OV-3 1.72E−05 2.74E−05 2.48E−05 >1.00E−04   1.68E−05 >1.00E−04  4.99E−05 3.95E−05 4.42E−05 Renal Cancer 786-0 1.06E−05 1.04E−051.61E−05 >1.00E−04   1.33E−05 5.03E−06 3.09E−05 1.76E−05 3.96E−05 A4982.86E−05 >1.00E−04   ACHN 1.81E−05 1.45E−05 2.88E−05 >1.00E−04  2.25E−05 >1.00E−04   3.24E−05 9.79E−05 >1.00E−04   CAKI-1 1.78E−051.60E−05 2.28E−05 >1.00E−04   2.23E−05 >1.00E−04  2.80E−05 >1.00E−04   >1.00E−04   RXF 393 2.98E−06 3.75E−065.25E−06 >1.00E−04   8.66E−06 2.72E−06 2.75E−05 1.50E−05 1.51E−05 TK-101.95E−05 1.31E−05 2.39E−05 >1.00E−04   1.64E−05 4.58E−05 3.07E−057.82E−05 4.86E−05 UO-31 2.33E−05 2.92E−05 6.29E−05 >1.00E−04  3.48E−05 >1.00E−04   3.62E−05 >1.00E−04   >1.00E−04   Prostate CancerPC-3 1.51E−05 1.07E−05 1.11E−05 >1.00E−04   1.44E−05 1.24E−05 2.25E−053.10E−05 4.64E−05 DU-145 1.47E−05 1.07E−05 1.63E−05 >1.00E−04   2.57E−057.23E−05 2.78E−05 3.27E−05 >1.00E−04   Breast Cancer MCF7 1.20E−054.42E−06 1.41E−05 >1.00E−04   1.61E−05 4.23E−05 3.21E−05 5.52E−055.72E−05 NCI/ 2.23E−05 1.80E−06 1.69E−05 >1.00E−04   2.42E−05 1.89E−052.74E−05 >1.00E−04   >1.00E−04   ADRRES MDA-MB- 1.79E−05 2.26E−053.37E−05 >1.00E−04   2.18E−05 3.51E−05 3.23E−05 4.82E−05 3.20E−05231/ATCC HS 578T 1.28E−05 1.58E−05 8.46E−06 >1.00E−04   1.51E−057.36E−06 3.28E−05 6.44E−05 8.08E−05 MDA-MB- 1.91E−052.39E−05 >1.00E−04   >1.00E−04   3.72E−05 >1.00E−04  2.13E−05 >1.00E−04   3.39E−05 435 MDA-N 2.88E−053.10E−05 >1.00E−04   >1.00E−04   4.54E−05 >1.00E−04  2.09E−05 >1.00E−04   2.51E−05 BT-549 3.98E−05 3.32E−05 >1.00E−04  6.84E−05 >1.00E−04   2.46E−05 >1.00E−04   >1.00E−04  

Example 37

[0483] Compounds corresponding to general formula 15 was synthesizedusing the method described below. Reference numerals and letterscorrespond with those shown in FIG. 24. The synthesized compounds wereN-[4-[1-methyl-2-(2,4-diaminofuro[2,3-d]pyrimidin-5-yl)ethyl]benzyl]-L-glutamicacid 24i, and its regio-restricted E isomer 24j and Z isomer 24j.

[0484] Reagents and conditions used in the synthesis are as follows: a.DMF, r.t.; b. (i) Bu₃P/DMSO (ii) NaH (iii) ethyl-4-acetylbenzoate; c. 5%Pd—C/H₂, 45 psi, MeOH/CHCl₃/DMF; d. (i) 1N NaOH, DMSO/MeOH (ii) 1N H; e.iBuOCOCl, Et3N, Diethyl-L-glutamate; f. (i) 1N NaOH, DMSO/MeOH (ii) 1NHCl; g. same as d; h. same as e; i. same as f.

[0485] A Wittig type condensation of2,4-diamino-5-(chloromethyl)furo[2,3-d]pyrimidine 24a withethyl-4-acetyl-benzoate furnished the desired two-carbon bridged9-methylfuro[2,3-d]- pyrimidine nucleus present in the target compoundsas generally taught by Gangjee A. et al. presented at the 217 ACSNational Meeting, Anaheim, Calif., Mar. 21-25, 1999, Abstract No. MEDI049. The crucial intermediate 24a was readily obtained by the reactionof 2,6-diaminopyrimidin-4-one and dichloroacetone in DMF at roomtemperature as previously reported by Gangjee et al., J. Med. Chem.41:1263-1271 (1998). Wittig condensation was first carried out bydisplacing the chloride with 3 eq of tributylphosphine and followed by2.2 eq of sodium hydride in anhydrous dimethylsulfoxide to form a lesssterically hindered ylide. This ylide was immediately condensed with thecorresponding ethyl-4-acetylbenzoate and after methanolic workupafforded the desired olefin 24b in 42% yield with both E and Z isomers.This was the common intermediate for the target molecules. E and Zisomer were assigned by the characteristic 6-CH chemical shift at 7.52ppm for the E isomer, which is 0.85 ppm downfield from the Z isomer(6.67 ppm); this compares favorably with the previously reported transand cis products. However, during the synthetic process some methylester exchange with ethyl ester was detected and the separation ofexcess tributylphosphine and phosphine oxide was also problematic.Reducing the amount of tributylphosphine and NaH to 1.5 eq, and using anethanol workup instead of methanol, afforded the desired olefin in 65%yield (E:Z ratio 2:1) as determined by ¹H NMR. Catalytic hydrogenationof the olefinic double bond of 24b was carried out under optimizedconditions which included 3 eq (w/w) of 5% palladium on activated carbonas catalyst in methanol/chloroform/N,N-dimethylformamide (30:70:2), 45psi of hydrogen and 4.5 h. After column chromatographic separation andrecrystallization from methanol the desired 8,9-dihydro derivative 24cwas obtained in 75% yield. Saponification of 24c with aqueous sodiumhydroxide afforded the free acid 24d in 90% yield. Subsequent couplingof 24d with diethyl-L-glutamate went smoothly using isobutylchloroformate as coupling agent and afforded the desired coupled product24e in 85% yield. Final saponification with aqueous sodium hydroxide atroom temperature followed by acidification to pH 4 in an ice bathafforded the pure target diacid 24i in 97% yield. The structure of 24iand the intermediates were confirmed by ¹H NMR and elemental analysis.

[0486] The isolation of the conformational restricted E and Z isomerstarted from the E and Z mixture 24b from the Wittig reaction. It wasmore efficient to separate the E and Z mixture after coupling with theL-glutamate diester than prior to coupling. Thus hydrolysis of 24b withaqueous sodium hydroxide afforded the E, Z free acids 24f in 92% yield.Subsequent coupling of 24f with diethyl-L-glutamate went smoothly usingisobutyl chloroformate as the coupling agent and afforded the desiredcoupled products 24 g and 24h in 85% yield as an E, Z mixture.Separation of the E isomer 24 g from the Z isomer 24h was achieved byrecrystallization from the methanol/ethylacetate (1:1). The whitecrystals which separated were the pure E isomer as confirmed by highperformance TLC and by its ¹H NMR. The Z isomer remained in thefiltrate. Final saponification with aqueous sodium hydroxide at roomtemperature followed by acidification to pH 4 in an ice bath affordedthe pure target E-diacid 24j in 97%. The Z-diacid 24k obtained in 95%yield was contaminated with 20% of 24j as indicated by the ¹H NMR.

Example 38

[0487] Compounds 24i, 24j and 24k prepared according to Example 37 wereevaluated as inhibitors of L. casei DHFR, E. coli DHFR, T. gondii DHFRand human recombinant DHFR. Reference numbers again correspond with FIG.24. The inhibitory concentration (IC₅₀) values are listed in Table 1 andcompared with the previous reported values for methotrexate (MTX). Theanalogue 24i inhibited human recombinant DHFR with an IC₅₀ 0.42 μM. Thisinhibitory activity was about 20 fold less than that of MTX. The Zisomer 24k was five fold less potent than the reduced analogue 24i whilethe E isomer was only marginally active against human recombinant DHFRwith an IC₅₀ of 42 μM. These three compounds were also evaluated asinhibitors of L. casei TS, E. coli TS, rat TS and human recombinant TSand were found to be weak inhibitors of these enzymes.

[0488] The target compounds 24i, 34j and 24k were also evaluated asinhibitors of the growth of leukemia CCRF-CEM cells in culture duringcontinuous exposure (Table 32). The reduced compound 24i was highlycytotoxic and had an IC₅₀=29 nM which was similar to MTX. Interestingly,in the transport deficient cell line R₂(Bos) compound 24i was about fourtimes more potent than MTX indicating-that transport is much more-important for MfX than 24i. The 7.5-fold decrease in activity of 24i inthe FPGS deficient cell-line indicates that polyglutamylation of 24idoes play a role in its cytotoxic activity. Again the pure E isomer wasessentially inactive with IC₅₀=340 μM and the 80% Z isomer mixture(IC₅=12 μM) was much less potent than 24i. TABLE 32 Growth Inhibition bycompounds 24i-24k and MTX of CCRF-CEM Human Leukemia Cells, ItsMethotrexate-resistant Sublines, Human Squamous Cell Carcinoma FaDu andA253 Cells During Continuous Exposure (0-120 h) Resistance EC₅₀(nM) Cellline mechanism MTX 24i 24j 24k CCRF- Sensitive  14.4 ± 1.0 29.2 ± 3.334000 ± 6000  1175 ± 83 CEM (n = 5) (n = 5) (n = 2) (n = 4) R1 Incr  675 ± 35  685 ± 45 nd 32000 ± 1000 DHFR (n = 2) (n = 2) (n = 2)R2(Bos) Def  1600 ± 100  430 ± 60 nd 17000 ± 0 Transport (n = 2) (n = 2)(n = 2) R30dm Decr   14 ± 0  220 ± 20 nd  6400 ± 900 FPGS (n = 2) (n =2) (n = 2) FaDu Sensitive  11.3 ± 1.8 17.5 ± 0.5 >10000  1400 ± 100 (n =2) (n = 2) (n = 2) (n = 2) A253 Sensitive  14.5 ± 0.5 28.5 ± 2.5 >10000 700 ± 70 (n = 2) (n = 2) (n = 2) (n = 2)

[0489] It will be appreciated by those skilled in the art that thepresent invention provides compounds, and pharmaceutically acceptablesalts thereof, effective against infections caused by Pneumocystiscarinii, Toxoplasmosis gondii, and other organisms, methods of preparingthese compounds, and methods of using these compounds in a patient fortherapeutic or prophylactic purposes. It will be further appreciated bythose skilled in the art that this invention provides compounds, andpharmaceutically acceptable salts thereof, effective in reducing tumorsor otherwise destroying cancerous cells in patients with cancer, methodsof preparing these compounds, and methods of using these compounds in apatient for therapeutic purposes.

[0490] Whereas particular embodiments of this invention have beendescribed above for purposes of illustration, it will be evident tothose skilled in the art that numerous variations of the details of thepresent invention may be made without departing from the invention asdefined in the appended claims.

What is claimed is:
 1. A compound, and pharmaceutically acceptablesalts, formulations, and prodrugs thereof, having the formula

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;wherein Z₄ and Z₅ are different and are selected from the groupconsisting of R₁₄ and

 where Z₄ is R₁₄ when Z₅ is

 and Z₄ is

 when Z₅ is R₁₄; wherein A₁ is CH; wherein B₁ is CH; wherein R₁₃ isselected from the group consisting of H, a lower alkyl group, asubstituted or unsubstituted aralkyl group, and a substituted orunsubstituted aryl group; wherein R₁₄ is selected from the groupconsisting of H, and a lower alkyl group; wherein R₁₅ is selected fromthe group consisting of H and a lower alkyl group; wherein R₁₆ isselected from the group consisting of H and a lower alkyl group; whereinR₁₇ is selected from the group consisting of aryl, diaryl, triaryl,mono-, di- or tri-substituted aryl, mono-, di- or tri-substituteddiaryl, mono-, di- or tri-substituted triaryl, a substituted orunsubstituted heteroaryl and p-aroyl-L-glutamate and each of saidsubstituents is independently selected from the group consisting of asubstituted or unsubstituted lower alkyl group, a substituted orunsubstituted alkoxy, a substituted or unsubstituted alkoxyaryloxy groupand a halogen; wherein R₁₈ is a lower alkyl group; and wherein eachlower alkyl group is independently selected from the group consisting oflower alkyl groups having from about 1 to 6 carbons.
 2. The compound ofclaim 1, wherein R₁₃ is selected from the group consisting of a benzylgroup and hydrogen and wherein R₁₇ is selected from the group consistingof phenyl, methoxyphenyl, trimethoxyphenyl, and p-benzoyl-L-glutamate.3. The compound of claim 2, wherein said methoxyphenyl is selected fromthe group consisting of 4-methoxyphenyl, 3-methoxyphenyl and2-methoxyphenyl and said trimethoxyphenyl is 3,4,5-trimethoxyphenyl. 4.The compound of claim 3 wherein R₁₃ is benzyl and R₁₇ is3-methoxyphenyl.
 5. The compound of claim 3 wherein R₁₃ is H and R₁₇ is2-methoxyphenyl.
 6. The compound of claim 3 wherein R₁₃ is benzyl andR₁₇ is 3,4,5-trimethoxyphenyl.
 7. A compound, and pharmaceuticallyacceptable salts, formulations and prodrugs thereof, having the formula

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;wherein Z₆ and Z₇ are different and are selected from the groupconsisting of R₁₄ and

 where Z₆ is R₁₄ when Z₇ is

 and

 when Z₅ is R₁₄; wherein A₂ is selected from the group consisting ofcarbon and CH; wherein B₂ is selected from the group consisting ofcarbon and CH; wherein the chemical bond between A₂ and B₂ is selectedfrom the group consisting of a single bond and a double bond, A₂ and B₂are carbon when the bond is a double bond and A₂ and B₂ are CH when thebond is a single bond; wherein R₁₄ is selected from the group consistingof H and a lower alkyl group; wherein R₁₅ is selected from the groupconsisting of H and a lower alkyl group; wherein R₁₆ is selected fromthe group consisting of H and a lower alkyl group; wherein R₁₇ isselected from the group consisting of aryl, diaryl, triaryl, mono-di ortri-substituted aryl, mono-, di- or tri-substituted diaryl, mono-, di-or tri-substituted triaryl, substituted or unsubstituted heteroaryl andp-aroyl-L-glutamate and each of said substituents is independentlyselected from the group consisting of a substituted or unsubstitutedlower alkyl group, a substituted or unsubstituted alkoxy, a substitutedor unsubstituted alkoxyaryloxy group, and a halogen; and wherein eachlower alkyl group is independently selected from the group consisting oflower alkyl groups having from about 1 to 6 carbons.
 8. The compound ofclaim 7, wherein L and M are both carbon, A₂ and B₂ are both carbon, andR₁₇ is selected from the group consisting of p-benzoyl-L-glutamate,phenyl, biphenyl, methoxyphenyl, dimethoxyphenyl, trimethoxyphenyl,chlorophenyl, dichlorophenyl, trichlorophenyl, naphthyl,methoxynaphthyl, and carbethoxyl phenyl.
 9. The compound of claim 8,wherein said methoxyphenyl is selected from the group consisting of2-methoxyphenyl and 3-methoxyphenyl; said dimethoxyphenyl is selectedfrom the group consisting of 3,4-dimethoxyphenyl and2,5-dimethoxyphenyl; said trimethoxyphenyl is 3,4,5-trimethoxyphenyl;said chlorophenyl is selected from the group consisting of2-chlorophenyl, 3-chlorophenyl and 4-chlorophenyl; said dichlorophenylis selected from the group consisting of 3,4-dichlorophenyl,2,5-dichlorophenyl, 2,4-dichlorophenyl; said trichlorophenyl is2,3,4-tricholorophenyl; said methoxynaphthyl is 6-methoxynaphthyl andsaid carbethoxy phenyl is 4carbethoxy phenyl.
 10. A method of treating apatient for an illness comprising the steps of: a) incorporating into asuitable pharmaceutical carrier a compound, or pharmaceuticallyacceptable salts, formulations and prodrugs thereof, having the formula:

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;wherein Z₄ and Z₅ are different and are selected from the groupconsisting of R₁₄ and

 where Z₄ is R₁₄ when Z₅ is

 and Z₄ is

 when Z₅ is R₁₄; wherein A₁ is CH; wherein B₁ is CH; wherein R₁₃ isselected from the group consisting of H, a lower alkyl group, asubstituted or unsubstituted aralkyl group, and a substituted orunsubstituted aryl group; wherein R₁₄ is selected from the groupconsisting of H, and a lower alkyl group; wherein R₁₅ is selected fromthe group consisting of H and a lower alkyl group; wherein R₁₆ isselected from the group consisting of H and a lower alkyl group; whereinR₁₇ is selected from the group consisting of aryl, diaryl, triaryl,mono-, di- or tri-substituted aryl, mono-, di or tri-substituted diaryl,mono-, di- or tri-substituted triaryl, a substituted or unsubstitutedheteroaryl and p-aryl-L-glutamate and each of said substituents isindependently selected from the group consisting of a substituted orunsubstituted lower alkyl group, a substituted or unsubstituted alkoxy,a substituted or unsubstituted alkoxyaryloxy group and a halogen;wherein R₁₈ is a lower alkyl group; and wherein each lower alkyl groupis selected from the group consisting of lower alkyl groups having fromabout 1 to 6 carbons; and b) administering an effective amount of saidcompound incorporated in said carrier to a patient.
 11. The method ofclaim 10, wherein R₁₃ is selected from the group consisting of a benzylgroup and hydrogen and wherein R₁₇ is selected from the group consistingof phenyl, methoxyphenyl, trimethoxyphenyl, and p-benzoyl-L, glutamate.13. The compound of claim 3 wherein R₁₃ is benzyl and R₁₇ is3-methoxyphenyl.
 14. The compound of claim 3 wherein R₁₃ is H and R₁₇ is2-methoxyphenyl.
 15. The compound of claim 3 wherein R₁₃ is benzyl andR₁₇ is 3,4,5-trimethoxyphenyl.
 16. The method of claim 10, wherein thetreatment is therapeutic and said illness is cancer.
 17. The method ofclaim 10, wherein the treatment is therapeutic and said illness isselected from the group consisting of infection caused by Pneumocystiscarinii and Toxoplasmosis gondii.
 18. The method of claim 10, whereinthe treatment is prophylactic and said illness is selected from thegroup consisting of infection caused by Pneumocystis carinii andToxoplasmosis gondii.
 19. The method of claim 10, wherein said carrieris selected from the group consisting of physiologic saline and 5%dextrose for injection.
 20. The method of claim 10, includingadministering said compound by a method selected from the groupconsisting of parenteral administration, oral administration and topicaladministration.
 21. A method of treating a patient comprising the stepsof: a) incorporating into a suitable pharmaceutical carrier a compound,or pharmaceutically acceptable salts, formulations and prodrugs thereof,having the formula:

wherein L and M are selected from the group consisting of carbon and CH,the chemical bond between L and M is selected from the group consistingof a single bond and a double bond, L and M are carbon when the bond isa double bond, and L and M are CH when the bond is a single bond;wherein Z₆ and Z₇ are different and are selected from the groupconsisting of R₁₄ and

 where Z₆ is R₁₄ when Z₇ is

 and Z₆ is

 when Z₅ is R₁₄; wherein A₂ is selected from the group consisting ofcarbon and CH; wherein B₂ is selected from the group consisting ofcarbon and CH; wherein the chemical bond between A₂ and B₂ is selectedfrom the group consisting of a single bond and a double bond, A₂ and B₂are carbon when the bond is a double bond and A₂ and B₂ are CH when thebond is a single bond; wherein R₁₄ is selected from the group consistingof H and a lower alkyl group; wherein R₁₅ is selected from the groupconsisting of H and a lower alkyl group; wherein R₁₆ is selected fromthe group consisting of H and a lower alkyl group; wherein R₁₇ isselected from the group consisting of aryl, diaryl, triaryl, mono-di ortri-substituted aryl, mono-, di- or tri-substituted diaryl, mono-, di-or tri-substituted triaryl, substituted or unsubstituted heteroaryl andp-aroyl-L-glutamate and each of said substituents is independentlyselected from the group consisting of a substituted or unsubstitutedlower alkyl group, a substituted or unsubstituted alkoxy, a substitutedor unsubstituted alkoxyaryloxy group, and a halogen; and wherein eachlower alkyl group is independently selected from the group consisting oflower alkyl groups having from about 1 to 6 carbons. b) administering aneffective amount of said compound incorporated in said carrier to apatient.
 22. The method of claim 19, wherein L and M are both carbon, A₂and B₂ are both carbon, and R₁₇ is selected from the group consisting ofp-benzoyl-L-glutamate, phenyl, biphenyl, methoxyphenyl, dimethoxyphenyl,trimethoxyphenyl, chlorophenyl, dichlorophenyl, trichlorophenyl,naphthyl, methoxynaphthyl, and carbethoxyl phenyl.
 23. The method ofclaim 20, wherein said methoxyphenyl is selected from the groupconsisting of 2-methoxyphenyl and 3-methoxyphenyl; said dimethoxyphenylis selected from the group consisting of 3,4-dimethoxyphenyl and2,5-dimethoxyphenyl; said trimethoxyphenyl is 3,4,5-trimethoxyphenyl;said chlorophenyl is selected from the group consisting of2-chlorophenyl, 3-chlorophenyl and 4-chlorophenyl; said dichlorophenylis selected from the group consisting of 3,4-dichlorophenyl,2,5-dimethoxyphenyl, 2,4-dichlorophenyl; said trichlorophenyl is2,3,4-tricholorophenyl; said methoxynaphthyl is 6-methoxynaphthyl andsaid carbethoxy phenyl is 4-carbethoxy phenyl.
 24. The method of claim19, wherein the treatment is therapeutic and said illness is cancer. 25.The method of claim 19, wherein the treatment is therapeutic and saidillness is selected from the group consisting of infection caused byPneumocystis carinii and Toxoplasmosis gondii.
 26. The method of claim19, wherein the treatment is prophylactic and said illness is selectedfrom the group consisting of infection caused by Pneumocystis cariniiand Toxoplasmosis gondii.
 27. The method of claim 19, wherein saidcarrier is selected from the group consisting of physiologic saline and5% dextrose for injection.
 28. The method of claim 19, includingadministering said compound by a method selected from the groupconsisting of parenteral administration, oral administration and topicaladministration.